Elimination of proliferating cells from stem cell-derived grafts

ABSTRACT

Provided herein are methods and compositions for a suicide gene approach comprising an expression vector comprising a cell cycle-dependent promoter driving the expression of a suicide gene. Also provided herein are methods to render proliferative cells sensitive to a prodrug after transplantation but avoids expression of the suicide gene in post-mitotic cells, such as neurons.

This application is a continuation of U.S. application Ser. No.16/204,320, filed Nov. 29, 2018, which claims the benefit of U.S.Provisional Patent Application No. 62/592,219, filed Nov. 29, 2017, eachof which is incorporated herein by reference in its entirety.

INCORPORATION OF SEQUENCE LISTING

This application contains a Sequence Listing XML, which has beensubmitted electronically and is hereby incorporated by reference in itsentirety. Said XML Sequence Listing, created on Nov. 30, 2022, is namedCLFRP0473USC1.xml and is 41,576 bytes in size.

BACKGROUND 1. Field

The present invention relates generally to the field of stem celltherapy. More particularly, it concerns the specific elimination ofproliferating cells in stem cell-derived grafts.

2. Description of Related Art

Parkinson's disease (PD) is a neurodegenerative disorder characterizedby the loss of the nigrostriatal pathway. Although the cause ofParkinson's disease is not known, it is associated with the progressivedegeneration or death of dopaminergic (i.e., tyrosine hydroxylase (TH)positive) neurons in the substantia nigra region of the basal ganglia,which induces progressive deterioration of motor function control. Thecharacteristic symptoms of Parkinson's disease appear when up to 70% ofTH-positive nigrostriatal neurons have degenerated.

There is currently no satisfactory cure for Parkinson's disease.Symptomatic treatment of the disease-associated motor impairmentsinvolves oral administration of dihydroxyphenylalanine (L-DOPA). L-DOPAis transported across the blood-brain barrier and converted to dopamine,partly by residual dopaminergic neurons, leading to a substantialimprovement of motor function. However, after a few years, thedegeneration of dopaminergic neurons progresses, the effects of L-DOPAare reduced and symptoms reappear.

Deep brain stimulation (DBS) therapy is the current preferred treatmentfor Parkinson's disease. DBS is a treatment of Parkinson's disease thataims to change the rates and patterns of activity of brain cells byimplanting a brain stimulator (i.e., an electrode) into a target regionin the brain known to be associated with movement, such as thesubthalamic nucleus, basal ganglia structures, including the globuspallidus internalis, or ventrointermediate nucleus of the thalamus.However, the success of DBS procedures can diminish over time.Therefore, better therapy for Parkinson's disease is necessary.

A treatment method for Parkinson's disease currently in developmentinvolves the transplantation of partially differentiated neuroepithelialcells (i.e., neural progenitor cells) into brain regions lackingsufficient dopaminergic signaling; these cells then differentiate intodopaminergic neurons in vivo. The potential of neural progenitor cellsto differentiate into dopaminergic neurons was demonstrated both withrodent and human PSC (Kriks et al., 2011; Wernig et al., 2008).Moreover, the potential of PSC-derived neural progenitor cells toreinnervate the striatum in drug-lesioned rodent and primate models ofParkinson's disease was shown (Ganat et al., 2012; Kriks et al., 2011;Wernig et al., 2008).

However, not all transplanted neural progenitor cells differentiate intothe desired neuronal subtypes and instead remain as proliferating cellswithin the graft, which leads to overgrowth and tumor formation. Eventhe most advanced protocols available (Ganat et al., 2012) have beenunable to overcome the problem of residual proliferating cells formingneuroepithelial tumors during PSC-derived neural progenitor cell-basedtherapy of CNS diseases. Therefore, there is an unmet need for methodsof eliminating residual proliferating cells following stem cell-basedtherapy.

SUMMARY

In certain embodiments, the present disclosure concerns isolated andrecombinant polynucleotides, such as polynucleotides comprising acell-cycle dependent promoter operatively linked to a suicide gene. Inone embodiment, there is provided an expression vector comprising a cellcycle-dependent promoter operatively linked to a suicide gene codingsequence, wherein the suicide gene is cytomegalovirus (CMV) UL97 ormutant HSV thymidine kinase (TK). In some aspects, the CMV-UL97 gene isa mutated version of wild-type CMV-UL97 comprising one or more aminoacid substitutions. The HSV-TK mutant can have one or more amino acidsubstitutions at residues 159-161 and 168-169 of HSV TK. The TK mutantused in the present methods may be SR11, SR26, SR39, SR4, SR15, SR32, orSR53 TK mutants. For example, the HSV TK can be HSV1-SR39TK (i.e,₁₅₉IFL₁₆₁ and ₁₆₈FM₁₆₉, amino acid substitution at position 159 isleucine to isoleucine, at position 160 is isoleucine to phenylalanine,at position 161 is phenylalanine to leucine, at position 168 is alanineto phenylalanine, and at position 169 is leucine to methionine).

In some aspects, the cell cycle-dependent promoter is a Ki-67, PCNA(proliferating cell nuclear antigen), CCNA2 (Cyclin A2), CCNB2 (CyclinB2), DLGAPS (DLG associated protein 5), or TOP2A (DNA topisomerase IIalpha) promoter. In some aspects, the cell cycle-dependent promoter is ahybrid of Ki-67, PCNA, CCNA2, CCNB2, DLGAPS, or TOP2A promoters. Inparticular aspects, the cell cycle-dependent promoter is a Ki-67promoter.

In some aspects, the expression vector further comprises a selectablemarker. In certain aspects, the selectable marker is an antibioticresistance gene or a gene encoding a fluorescent protein.

In certain aspects, the expression vector is further defined as a viralvector. In some aspects, the viral vector is a lentiviral vector, anadenoviral vector, a retroviral vector, a vaccinia viral vector, anadeno-associated viral vector, a herpes viral vector, or a polyoma viralvector. In particular aspects, the viral vector is a lentiviral vector.

In another embodiment, there is provided a host cell comprising thepolynucleotide comprising a cell-cycle dependent promoter operativelylinked to a suicide gene. In particular aspects, the host cell isfurther defined as a precursor cell. In some aspects, the polynucleotideis introduced into the host cell using a viral vector, such as alentiviral vector, retroviral vector, an adenoviral vector (e.g., anintegrating adenovector), a vaccinia viral vector, an adeno-associatedviral vector, a herpes viral vector, or a polyoma viral vector. Inparticular aspects, the viral vector is a lentiviral vector. The vectormay be an integrating vector. In other aspects, the polynucleotide isintroduced into the host cell using a non-viral approach, such asinjection of naked DNA, nucleic acid delivery enhanced by physicalmethods (e.g., electroporation or gene gun), or nucleic acid deliveryenhanced by chemical methods (e.g., lipids, liposomes nanoparticles, orcell permeating peptides).

In some aspects, the host cell is further defined as a neural precursorcell, cardiomyocyte precursor cell, endothelial precursor cell,pancreatic precursor cell, kidney precursor cell, oligodendrocyteprecursor cell, hematopoietic precursor cell, myeloid precursor cell,mesenchymal precursor cell, retinal precursor cell, or osteoclastprecursor cell. In particular aspects, the host cell is further definedas a neural precursor cell. In some aspects, the cell is derived from apluripotent stem cell (PSC). In some aspects, the PSC is an inducedpluripotent stem cell (iPS cell) or embryonic stem cell (ESC). Inparticular aspects, the neural precursor cell is further defined as acell expressing at least one of the markers selected from the groupconsisting of musashi, nestin, sox2, vimentin, pax6, and sox1. Incertain aspects, the neural precursor cell expresses 2, 3, 4, 5, or all6 of the markers musashi, nestin, sox2, vimentin, pax6, and sox1.

In a further embodiment, there is provided a host cell comprising anexpression vector of the embodiments. In some aspects, the host cell isfurther defined as a neural precursor cell, cardiomyocyte precursorcell, endothelial precursor cell, pancreatic precursor cell, kidneyprecursor cell, oligodendrocyte precursor cell, hematopoietic precursorcell, myeloid precursor cell, mesenchymal precursor cell, retinalprecursor cell, or osteoclast precursor cell. In particular aspects, thehost cell is further defined as a neural precursor cell. In someaspects, the cell is derived from a pluripotent stem cell (PSC). In someaspects, the PSC is an induced pluripotent stem cell (iPS cell) orembryonic stem cell (ESC). In particular aspects, the neural precursorcell is further defined as a cell expressing at least one of the markersselected from the group consisting of musashi, nestin, sox2, vimentin,pax6, and sox1. In certain aspects, the neural precursor cell expresses2, 3, 4, 5, or all 6 of the markers musashi, nestin, sox2, vimentin,pax6, and sox1.

In an even further embodiment, there is provided a pharmaceuticalcomposition comprising the host cell of the embodiments and, optionally,a pharmaceutically acceptable carrier.

In another embodiment, there is provided a method of producing hostcells (e.g., precursor cells) of the embodiments comprising obtaining astarting population of PSCs; differentiating the PSCs into precursorcells; and isolating and culturing the precursor cells, wherein eitherthe PSCs or the precursor cells are transfected or transduced with apolynucleotide comprising a cell-cycle dependent promoter operativelylinked to a suicide gene (e.g, in an expression vector of theembodiments) and selected for the presence of said polynucleotide (e.g.,the expression vector), such that the cultured precursor cells comprisethe cell cycle-dependent promoter operatively linked to the suicide genecoding sequence.

In some aspects, the precursor cells are neural precursor cells,cardiomyocyte precursor cells, endothelial precursor cells, pancreaticprecursor cells, kidney precursor cells, oligodendrocyte precursorcells, hematopoietic precursor cells, myeloid precursor cells,mesenchymal precursor cells, retinal precursor cells, or osteoclastprecursor cells. In certain aspects, the precursor cells are neuralprecursor cells.

In certain aspects, the pluripotent stem cell population is an embryonicstem cell. In particular aspects, the pluripotent stem cell populationis an induced pluripotent stem cell population.

In some aspects, differentiating cells of the population into neuralprecursor cells comprises contacting the pluripotent stem cellpopulation with fibroblast growth factor or epidermal growth factor.

In certain aspects, the method further comprises contacting thepluripotent stem cell population with N2 and B27. In some aspects,isolating comprises sorting the cells of the population to isolateprecursor cells, such as neural progenitor cells.

In some aspects, cells are selected for the presence of said expressionvector by a method comprising contacting the transfected or transducedcells with an antibiotic. In certain aspects, cells are selected for thepresence of said expression vector by a method comprising sorting thetransfected or transduced cells.

A further embodiment provides a method of cell replacement therapy forreplacing cells that are known to be essentially non-dividing cells, themethod comprising administering an effective amount of host cells (e.g.,precursor cells) of the embodiments, and administering to the subject anamount of a prodrug that is activated by the suicide gene, the prodrugbeing administered in an amount effective to eliminate cycling precursorcells In some aspects, the subject is a mammal. In certain aspects, themammal is a mouse, rat, non-human primate, or human. In some aspects,the genome of the host cell comprises a genome essentially identical tothe genome of the subject and an expression vector of the embodiments.In some aspects, the essentially non-dividing cells to be replacedcomprise dopaminergic cells and the precursor cell population comprisesdopaminergic neural precursor cells (e.g., defined as expressingtyrosine hydroxylase or dopamine active transporter). In certainaspects, the subject has Parkinson's disease. In particular aspects, theprodrug is ganciclovir and/or acyclovir. In specific aspects, theprodrug is penciclovir In some aspects, the prodrug is administered morethan once. In some aspects, the prodrug is administered after asufficient period of time for the precursor cells to initiatedifferentiation. In some aspects, the period of time is 3-6 days, suchas 3, 4, 5, or 6 days after administering the precursor cells. In otheraspects, the period of time is 7-15 days, such as 7, 8, 9, 10, 11, 12,13, 14, or 15 days after administering the precursor cells. In otheraspects, the precursor cells and pro-drug are administered concurrently.In some aspects, the prodrug is administered by injection

Other objects, features, and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings(s) will be provided by the Office upon request andpayment of the necessary fee.

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIGS. 1A-1C: Elaboration of a pluripotent stem cell line expressing asuicide gene under the control of a cell cycle-dependent promoter. (A)Thymidine kinase from herpes simplex virus (HSV-TK) was introduced intoa lentiviral vector under the control of a Ki67 promotor fragment. TheTK used in this study was a fusion protein containing a C-terminallyfused zeocin resistance gene. The construct was transduced into theclinical grade human pluripotent stem cell line HS415 (TK-PSC). TK-PSCcells expressing this enzyme were selected by their resistance tozeocin. (B) Cell preparations used in the present study: pluripotentstem cells (TK-PSC, left panel) were differentiated into neurospherescontaining dopaminergic neuroprogenitors (TK-NPC, middle panel) andterminated maturation into dopaminergic neurons (TH staining ofTK-neurons, right panel). (C) Characterization of TK-PSC cells inundifferentiated state (upper panel) and upon differentiation intoneurons (lower panel). Ki67 and HSV-TK were detected by immunostainingand nucleus by DAPI. Both TK-PSC and TK-Neuron showed staining for Ki67and HSV-TK which overlap in the merged images.

FIGS. 2A-2D: Effect of ganciclovir treatment in vitro. (A) Analysis ofexpression of proliferation markers (Ki67) and pluripotency markers(nanog, oct3/4) at different stages of the differentiation protocoldescribed in FIG. 1B. Protein expression was analyzed by flow cytometry(mean+/−SEM of 4 independent experiments). (B) a-d: Undifferentiatedpluripotent TK-PSC cells were exposed to increasing concentrations ofganciclovir (0, 2.5, 5, and 10 μM). (B) e-f: comparison of the effect of40 μM ganciclovir on TK-expressing and control PSC. Note that in controlPSC, no ganciclovir toxicity is observed even with the highestconcentration of ganciclovir (40 μM). Upon neuronal differentiation,TK-expressing cells lose their sensitivity to ganciclovir as predictedby the lack of Ki67-driven TK expression in post-mitotic neurons (seeFIG. 1C). (C) Dose response to ganciclovir: TK-PSC, control PSC;TK-neurons, and control neurons were exposed to increasingconcentrations of ganciclovir. Cell viability was monitored usingcalcein. The control PSCs had higher expression of Calcein as comparedto the TK-PSCs at increasing concentrations of ganciclovir. (D)Ganciclovir time course: TK and control PSC, 1 week NPC (TK andcontrol), and 2 week NPC (TK and control) were exposed to 40 μMganciclovir and cell toxicity was monitored using calcein. Data frompanel C and D are shown as triplicate determinations and arerepresentative of 3 independent experiments. Error bars=+/−SD, n=3.

FIGS. 3A-3C: Schedule of cell transplantation and ganciclovir treatment.Different transplantation and ganciclovir treatment protocols were used.(A) Schematic representation of the differentiation protocol towards DAneurons. Pluripotent stem cells were cultured as neurospheres for 1 weekfor midbrain orientation phase, followed by a 2 or 3 week-maturationphase. (B) Transplantation of undifferentiated pluripotent stem cellswith early or late ganciclovir treatment. Early treatment:undifferentiated pluripotent stem cells were transplanted andganciclovir (or PBS control) was applied by daily intra-peritonealinjection from day 4 to day 19 post-transplantation. Late treatment:undifferentiated pluripotent stem cells were transplanted andganciclovir (or PBS control) was applied by daily intra-peritonealinjection from day 30 to day 45 post-transplantation. (C)Transplantation of 2 or 3 week NPC: NPC containing—neurospheres weredissociated and transplanted into mice striatum. Ganciclovir (or PBScontrol) treatment was given from day 4 to day 19 post-transplantation.For all protocols, animals were sacrificed 1 month after termination ofganciclovir treatment.

FIGS. 4A-4B: Early ganciclovir treatment prevents tumor formation aftertransplantation of HSV-TK-expressing pluripotent cells. (A)Transplantation of TK-PSC without ganciclovir treatment: teratomaformation was consistently observed after transplantation of TK-PSC intoPBS-treated mice top panel: cresyl violet coloration; a-d lower panels:immunostaining of the graft (HCM staining allows detection of humantypical proteins) show a development mainly towards neural tissue withexpression of nestin (for immature neural cells), beta III tubulin (formature neurons). Proliferative cells are stained with Ki67 and PCNA. (B)Transplantation of TK-PSC with early ganciclovir treatment: absence ofteratoma formation and cell proliferation in mice transplanted withTK-PSC followed by early ganciclovir treatment (day 4 to 19 followingtransplantation); a-d top panel: cresyl violet coloration; e-f lowerpanels: immunostaining with HCM, Ki67 and PCNA. Mice were sacrificed andimmunohistochemistry was performed one month after termination ofganciclovir treatment. Stainings shown in this figure are representativeof 3-5 mice per group. Cells stained positive for Ki67 and HCM or PCNA;the staining overlaps in the merged images.

FIGS. 5A-5B: Early ganciclovir treatment and impact on Iba1 andKi67-positive cells. (A) In the absence of ganciclovir treatment, therewere abundant HSV-TK-containing, proliferating (Ki67), human (HCM) tumorcells; the tumors were infiltrated by microglia (Iba1). (B) Inganciclovir-treated mice, a human cell graft (HCM) was barely detectableand virtually no Ki67 and TK expression was observed in the graftregion. Some microglia staining by Iba1 was detectable around the scarof the graft. Mice were sacrificed and immunohistochemistry wasperformed one month after termination of ganciclovir treatment.

FIGS. 6A-6B: Late ganciclovir treatment does not prevent tumor formationafter transplantation of HSV-TK-expressing pluripotent cells. Mice weretransplanted with TK-PSC and PBS-treatment (A) or ganciclovir-treatment(B) was initiated one month after transplantation. Treatment wasmaintained for 15 days, followed by 1 month without treatment beforesacrifice. Under these conditions, there was no difference betweenPBS-treated and ganciclovir-treated mice. The grafts have developedtowards a tumor with predominantly neural tissue (beta III tubulinstaining). Tumor cells expressed Ki67 and TK, but no Oct3/4. The graftswere vascularized, as evidenced by CD31 staining for endothelial cells.

FIGS. 7A-7C: Sequencing of HSV-TK in pluripotent stem cell-derivedtumors subjected to late ganciclovir treatment. (A) 2 weeks ganciclovirtreatment was initiated 4 weeks after intrastriatal transplantation ofpluripotent stem cells and mice were sacrificed 4 weeks aftertermination of treatment (as described in FIG. 6 ). DNA was extractedfrom PFA-fixed, paraffin-embedded tumor samples and amplified using theindicated PCR primers. (B) Direct sequencing of plasmid DNA andamplified sequences from tumor samples did not detect any mutations.(Amplicon 1—plasmid=SEQ ID NO: 11; “seq”=SEQ ID NO: 12; Amplicon2—plasmid=SEQ ID NO: 13; “seq”=SEQ ID NO: 14; Amplicon 3—plasmid=SEQ IDNO: 15; “seq”=SEQ ID NO: 16; Amplicon 4—plasmid=SEQ ID NO: 17; “seq”=SEQID NO: 18; Amplicon 5—plasmid=SEQ ID NO: 19; “seq”=SEQ ID NO: 20) (C)Sequencing of HSV-TK subclones from amplicon 3 and 5 (derived from PCRreactions described for panel B). amplicon 3: plasmid DNA used for celltransduction, as well as tumor-derived cDNA clones did not contain thesplice donor site found in the HSV-TK reference sequence. However, cDNAclones 3 and 4 showed coding non-synonymous mutations. (ref=SEQ ID NO:21; plasmid=SEQ ID NO: 22; clone 1=SEQ ID NO: 23; clone 2=SEQ ID NO: 24;clone 3=SEQ ID NO: 25; clone 4=SEQ ID NO: 26; ATP-binding site=SEQ IDNO: 27; nucleotide-binding site=SEQ ID NO: 28) amplicon5: none of thecDNA clones showed any mutations. (plasmid=SEQ ID NO: 29; clone 1-7=SEQID NO: 30)

FIGS. 8A-8D: Graft development after transplantation of neural precursorcells (NPC). Mice were transplanted with NPC and treated with PBS organciclovir on days7-22 following transplantation. Mice were sacrificed4 weeks after the termination of ganciclovir treatment and brains wereanalyzed. There was no tumor formation, but the grafts had developedinto a tissue integrated into the mouse brain. No difference wasobserved between PBS and ganciclovir treatment (A) cresyl violetcoloration. Transplants were strongly positive for beta 3-tubulin (B)upper panel, and weekly positive for TH (B) lower panel. Transplantedcells were PCNA-positive, but Ki67 and BrdU negative (C and insert). (D)Size of transplants under different experimental conditions in theabsence (grey histogram) or presence (black histogram) of ganciclovir.Histograms on the left and middle show transplants after injection ofpluripotent stem cells (PSC) treated for 2 weeks with ganciclovir 5 days(early treatment) or 30 days after cell transplantation (latetreatment). Histograms on the right show transplants after injection ofNPC treated with ganciclovir, 5 days after transplantation. Errorbars=mean+/−SEM, n=3 to 5 in each group of mice. *p=0.0286 in MannWhitney test.

FIG. 9 : Image of plate in cell viability assay. Cells were transfectedwith a construct expressing HSV-TK, SR39, or CMV-UL97.

FIGS. 10A-10B: (A) Cell viability assay with cells transfected withHSV-TK or SR39. Cells were treated with acyclovir or ganciclovir. (B)Cell viability assay with cells transfected with HSV-TK or SR39 andtreated with penciclovir.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Pluripotent stem cell (PSC)-based cell therapy is an attractive concept,in particular for neurodegenerative diseases. However, transplantationof undifferentiated PSC or rapidly proliferating precursor cells canlead to tumor formation. Thus, to translate promising animal data into afuture clinical use, safety mechanisms to eliminate proliferating cellsare needed. Thus, in certain embodiments, the present disclosureprovides methods and compositions for a suicide gene approach toeliminate proliferating cells. In an exemplary method, an expressionvector is provided based on a cell cycle-dependent promoter (e.g., Ki67)driving the expression of a suicide gene (e.g., cytomegalovirus (CMV)UL97). The present studies show for the first time the use of theCMV-UL97 gene as a suicide gene. Thus, this construct provides methodsto render proliferative cells sensitive to ganciclovir aftertransplantation but avoids expression of the antigenic viral suicidegene protein in post-mitotic neurons.

The present disclosure shows that host cells comprising the exemplaryKi67-HSV-TK construct or CMV-UL97 killed proliferating PSC and earlyneural precursor cells (NPC) by exposure to ganciclovir, acyclovir, orpenciclovir in vitro. In addition, in vivo transplantation of PSCinduced a teratoma which was prevented by early (e.g., 4 dayspost-transplant) treatment with ganciclovir. Thus, the suicide geneapproach of the present disclosure allows killing of proliferatingundifferentiated and/or overgrowth of early precursor cells withoutexpression of the suicide gene in mature neurons. This approach has thepotential to be useful for other stem cell-based therapies, where thefinal target is a post-mitotic cell (e.g. cardiomyocytes, or pancreaticbeta cells).

I. DEFINITIONS

As used herein, “essentially free,” in terms of a specified component,is used herein to mean that none of the specified component has beenpurposefully formulated into a composition and/or is present only as acontaminant or in trace amounts. The total amount of the specifiedcomponent resulting from any unintended contamination of a compositionis therefore well below 0.05%, preferably below 0.01%. Most preferred isa composition in which no amount of the specified component can bedetected with standard analytical methods.

As used herein the specification, “a” or “an” may mean one or more. Asused herein in the claim(s), when used in conjunction with the word“comprising,” the words “a” or “an” may mean one or more than one.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.” As used herein “another”may mean at least a second or more.

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects.

The term “cell” is herein used in its broadest sense in the art andrefers to a living body that is a structural unit of tissue of amulticellular organism, is surrounded by a membrane structure thatisolates it from the outside, has the capability of self-replicating,and has genetic information and a mechanism for expressing it. Cellsused herein may be naturally-occurring cells or artificially modifiedcells (e.g., fusion cells, genetically modified cells, etc.).

The term “stem cell” refers herein to a cell that under suitableconditions is capable of differentiating into a diverse range ofspecialized cell types, while under other suitable conditions is capableof self-renewing and remaining in an essentially undifferentiatedpluripotent state. The term “stem cell” also encompasses a pluripotentcell, multipotent cell, precursor cell and progenitor cell. Exemplaryhuman stem cells can be obtained from hematopoietic or mesenchymal stemcells obtained from bone marrow tissue, embryonic stem cells obtainedfrom embryonic tissue, or embryonic germ cells obtained from genitaltissue of a fetus. Exemplary pluripotent stem cells can also be producedfrom somatic cells by reprogramming them to a pluripotent state by theexpression of certain transcription factors associated withpluripotency; these cells are called “induced pluripotent stem cells,”“iPS cells” or “iPSCs”.

An “embryonic stem (ES) cell” is an undifferentiated pluripotent stemcell which is obtained from an embryo in an early stage, such as theinner cell mass at the blastocyst stage, or produced by artificial means(e.g. nuclear transfer) and can give rise to any differentiated celltype in an embryo or an adult, including germ cells (e.g. sperm andeggs).

“Induced pluripotent stem cells (iPSCs)” are cells generated byreprogramming a somatic cell by expressing or inducing expression of acombination of factors (herein referred to as reprogramming factors).iPSCs can be generated using fetal, postnatal, newborn, juvenile, oradult somatic cells. In certain embodiments, factors that can be used toreprogram somatic cells to pluripotent stem cells include, for example,Oct4 (sometimes referred to as Oct 3/4), Sox2, c-Myc, Klf4, Nanog, andLin28. In some embodiments, somatic cells are reprogrammed by expressingat least two reprogramming factors, at least three reprogrammingfactors, or four reprogramming factors to reprogram a somatic cell to apluripotent stem cell.

“Pluripotent stem cell” refers to a stem cell that has the potential todifferentiate into all cells found in an organism preferably, cellsrepresenting any of the three germ layers: endoderm (e.g., interiorstomach lining, gastrointestinal tract, and the lungs), mesoderm (e.g.,muscle, bone, blood, and urogenital), or ectoderm (e.g., epidermaltissues and nervous system).

As used herein, the term “somatic cell” refers to any cell other thangerm cells, such as an egg or a sperm which does not directly transferits DNA to the next generation. Typically, somatic cells have limited orno pluripotency. Somatic cells used herein may be naturally-occurring orgenetically modified.

As used herein the term “engineered” in reference to cells refers tocells that comprise at least one genetic element exogenous to the cellthat is integrated into the cell genome. In some aspects, the exogenousgenetic element can be integrated at a random location in the cellgenome. In other aspects, the genetic element is integrated at aspecific site in the genome. For example, the genetic element may beintegrated at a specific position to replace an endogenous nucleic acidsequence, such as to provide a change relative to the endogenoussequence (e.g., a change in single nucleotide position).

A “precursor cell” as used herein refers to a stem cell which has thepotential to differentiate into many different (pluri- and multipotent)or two different (bipotent) mature cell types. A precursor cell may alsobe a stem cell which has the capacity to differentiate into only onecell type. For example, precursor cells include neural precursor cells(NPCs), cardiomyocyte precursor cells and pancreatic precursor cells.“Neural precursor cells” are defined herein as immature cells of thenervous system which have the potential to develop into mature nervoussystem cells such as neurons and glia (e.g., astrocytes andoligodendrocytes).

The term “suicide gene” refers to a gene whose protein product convertsa non-toxic prodrug into a toxic drug (e.g., an active chemotherapeuticagent), thereby killing cells that express the gene product.

As used herein, the term “polynucleotide” refers to a nucleic acidmolecule that either is recombinant or has been isolated free of totalgenomic nucleic acid. By “isolated” when referring to a nucleotidesequence, is meant that the indicated molecule is present in thesubstantial absence of other biological macromolecules of the same type.Included within the term “polynucleotide” are recombinant vectors,including, for example, plasmids, cosmids, phage, viruses, and the like.Polynucleotides include, in certain aspects, regulatory sequences,isolated substantially away from their naturally occurring genes orprotein coding sequences. Polynucleotides may be single-stranded (codingor antisense) or double-stranded, and may be RNA, DNA (e.g., genomicDNA, cDNA, or synthetic DNA), analogs thereof, or a combination thereof.Additional coding or non-coding sequences may, but need not, be presentwithin a polynucleotide.

The term “expression vector” refers to a vector containing a nucleicacid sequence coding for at least part of a gene product capable ofbeing transcribed. In some cases, RNA molecules are then translated intoa protein, polypeptide, or peptide. Expression vectors can contain avariety of “control sequences,” which refer to nucleic acid sequencesnecessary for the transcription and possibly translation of anoperatively linked coding sequence in a particular host organism,including, but not limited to, promoter regions, polyadenylationsignals, transcription termination sequences, upstream regulatorydomains, internal ribosome entry sites (IRES), enhancers, and the like.In addition to control sequences that govern transcription andtranslation, vectors and expression vectors may contain nucleic acidsequences that serve other functions as well, such as origins ofreplication for the replication of a vector in a recipient cell. Not allof these control elements need always be present so long as the selectedcoding sequence is capable of being replicated, transcribed, andtranslated in an appropriate host cell.

The term “promoter region” is used herein in its ordinary sense to referto a nucleotide region comprising a DNA regulatory sequence, wherein theregulatory sequence is derived from a gene that is capable of bindingRNA polymerase and other transcription factors and initiatingtranscription of a downstream coding sequence. Promoter regions alsocontrol the rate of transcription. A promoter may or may not be used inconjunction with an “enhancer,” which refers to a cis-acting regulatorysequence involved in the transcriptional activation of a nucleic acidsequence. “Operatively linked” refers to an arrangement of elementswherein the components so described are configured so as to performtheir usual function. Thus, control sequences operatively linked to acoding sequence are capable controlling the transcriptional initiationand expression of that sequence. The control elements need not becontiguous with the coding sequence, so long as they function to directthe expression thereof. Thus, for example, intervening untranslated yettranscribed sequences can be present between a promoter sequence and thecoding sequence and the promoter sequence can still be considered“operatively linked” to the coding sequence.

The term “heterologous,” as it relates to nucleic acid sequences, suchas gene sequences and control sequences, denotes sequences that are notnormally joined together and/or are not normally associated with aparticular cell. Thus, a “heterologous” region of a nucleic acidconstruct or a vector is a segment of nucleic acid within or attached toanother nucleic acid molecule that is not found in association with theother molecule in nature. For example, a heterologous region of anucleic acid construct could include a coding sequence flanked bysequences not found in association with the coding sequence in nature.Another example of a heterologous coding sequence is a construct wherethe coding sequence itself is not found in nature (e.g., syntheticsequences having codons different from the native gene). Similarly, acell transformed with a construct that is not normally present in thecell would be considered heterologous for purposes of the presentdisclosure. Allelic variation or naturally occurring mutational eventsdo not give rise to heterologous DNA, as used herein.

For the purpose of describing the relative position of nucleotidesequences in a particular nucleic acid molecule throughout the presentapplication, such as when a particular nucleotide sequence is describedas being situated “upstream,” “downstream,” “5′,” or “3′” relative toanother sequence, it is to be understood that it is the position of thesequences in the non-transcribed strand of a DNA molecule that is beingreferred to as is conventional in the art.

“Homology” refers to the percent identity between two polynucleotidemoieties. Two polynucleotide sequences are “substantially homologous” toeach other when the sequences exhibit at least about 50%, preferably atleast about 75%, more preferably at least about 80%-85%, preferably atleast about 90%, and most preferably at least about 95%-98% sequenceidentity over a defined length of the molecules. As used herein,substantially homologous also refers to sequences showing completeidentity to the specified polynucleotide sequence.

The term “transfection” is used to refer to the uptake of foreign DNA bya cell. A cell has been “transfected” when exogenous DNA has beenintroduced inside the cell membrane. A number of transfection techniquesare generally known in the art. See, e.g., Graham et al. (1973)Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratorymanual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986)Basic Methods in Molecular Biology, Elsevier, and Chu et al. Gene 13:197, 1981. Such techniques can be used to introduce one or moreexogenous DNA moieties, such as a plasmid vector and other nucleic acidmolecules, into suitable host cells. The term refers to both stable andtransient uptake of the genetic material.

The term “transduction” denotes the delivery of a DNA molecule to arecipient cell either in vivo or in vitro, via a replication-defectiveviral vector, such as via a recombinant lentiviral vector particle.

By “vertebrate subject” is meant any member of the subphylum chordata,including, without limitation, mammals such as cattle, sheep, pigs,goats, horses, and human and non-human primates; domestic animals suchas dogs and cats; laboratory animals including rodents such as mice,rats and guinea pigs, and the like; birds, including domestic, wild andgame birds such as cocks and hens including chickens, turkeys and othergallinaceous birds; and fish. The term does not denote a particular age.Thus, both adult and newborn animals, as well as fetuses, are intendedto be covered.

By “subject” or “patient” is meant any single subject for which therapyis desired, including humans, cattle, dogs, guinea pigs, rabbits,chickens, and so on. Also intended to be included as a subject are anysubjects involved in clinical research trials not showing any clinicalsign of disease, or subjects involved in epidemiological studies, orsubjects used as controls.

Within the context of the present disclosure, the term “thymidine kinasemutant” should be understood to include not only the specific proteindescribed herein (as well as the nucleic acid sequences which encodethese proteins), but derivatives thereof which may include variousstructural forms of the primary protein which retain biologicalactivity. For example, a thymidine kinase mutant may be in the form ofacidic or basic salts, or in neutral form. In addition, individual aminoacid residues may be modified by oxidation or reduction. Furthermore,various substitutions, deletions, or additions may be made to the aminoacid or nucleic acid sequences, the net effect of which is to retain orfurther enhance the increased biological activity of the mutant. Due tocode degeneracy, for example, there may be considerable variation innucleotide sequences encoding the same amino acid sequence.

II. CELLS OF THE PRESENT DISCLOSURE

In certain embodiments of the present disclosure, there are disclosedmethods and compositions for producing precursor cells, such as neuralprecursor cells, comprising a construct with a cell-cycle dependentpromoter operatively linked to a suicide gene, such as HSV-TK orCMV-UL97. In some aspects, the precursor cells are derived from astarting population of pluripotent stem cells (PSCs), such as embryonicstem cells or induced pluripotent stem cells.

A. Pluripotent Stem Cells

The starting population of PSCs of the present disclosure can be humanembryonic stem cells (ESCs) or induced pluripotent stem cells (iPSC).Both ESCs and iPSCs are capable of long-term proliferation in vitro,while retaining the potential to differentiate into all cell types ofthe body, including neural precursor cells, cardiomyocytes, pancreaticbeta cells, and hepatocytes. Certain aspects of the present disclosureconcern precursor cells that could be induced directly from human ESC oriPSCs via expression of a combination of transcription factors fordifferentiation/function, similar to the generation of iPSCs, bypassingmost, if not all, normal developmental stages.

1. Embryonic Stem Cells

In certain aspects, the precursor cells are derived from ESCs. ESCs arederived from the inner cell mass of blastocysts and have a high in vitrodifferentiating capability. ESCs can be isolated by removing the outertrophectoderm layer of a developing embryo, then culturing the innermass cells on a feeder layer of non-growing cells. The replated cellscan continue to proliferate and produce new colonies of ESCs which canbe removed, dissociated, replated again and allowed to grow. Thisprocess of “subculturing” undifferentiated ES cells can be repeated anumber of times to produce cell lines containing undifferentiated EScells (U.S. Pat. Nos. 5,843,780; 6,200,806; 7,029,913). ESCs have thepotential to proliferate while maintaining their pluripotency. Forexample, ESCs are useful in research on cells and on genes which controlcell differentiation. The pluripotency of ESCs combined with geneticmanipulation and selection can be used for gene analysis studies in vivovia the generation of transgenic, chimeric, and knockout mice.

Methods for producing mouse ESCs are well known. In one method, apreimplantation blastocyst from the 129 strain of mice is treated withmouse antiserum to remove the trophoectoderm, and the inner cell mass iscultured on a feeder cell layer of chemically inactivated mouseembryonic fibroblasts in medium containing fetal calf serum. Colonies ofundifferentiated ES cells that develop are subcultured on mouseembryonic fibroblast feeder layers in the presence of fetal calf serumto produce populations of ESCs. In some methods, mouse ESCs can be grownin the absence of a feeder layer by adding the cytokine leukemiainhibitory factor (LIF) to serum-containing culture medium (Smith,2000). In other methods, mouse ESCs can be grown in serum-free medium inthe presence of bone morphogenetic protein and LIF (Ying et al., 2003).

Human ESCs can be produced or derived from a zygote or blastocyst-stagedmammalian embryo produced by the fusion of a sperm and egg cell, nucleartransfer, pathogenesis, or the reprogramming of chromatin and subsequentincorporation of the reprogrammed chromatin into a plasma membrane toproduce an embryonic cell by previously described methods (Thomson andMarshall, 1998; Reubinoff et al., 2000). In one method, humanblastocysts are exposed to anti-human serum, and trophectoderm cells arelysed and removed from the inner cell mass which is cultured on a feederlayer of mouse embryonic fibroblasts. Further, clumps of cells derivedfrom the inner cell mass are chemically or mechanically dissociated,replated, and colonies with undifferentiated morphology are selected bymicropipette, dissociated, and replated. In some methods, human ESCs canbe grown without serum by culturing the ESCs on a feeder layer offibroblasts in the presence of basic fibroblast growth factor (Amit etal., 2000). In other methods, human ESCs can be grown without a feedercell layer by culturing the cells on a protein matrix such as MATRIGEL™or laminin in the presence of “conditioned” medium containing basicfibroblast growth factor (Xu et al., 2001).

ESCs can also be derived from other organisms including rhesus monkeyand marmoset by previously described methods (Thomson, and Marshall,1998; Thomson et al., 1995; Thomson and Odorico, 2000; U.S. Pat. No.5,843,780), as well as from established mouse and human cell lines. Forexample, established human ESC lines include MAOI, MA09, ACT-4, HI, H7,H9, H13, H14 and ACT30. As a further example, mouse ESC lines that havebeen established include the CGR8 cell line established from the innercell mass of the mouse strain 129 embryos, and cultures of CGR8 cellscan be grown in the presence of LIF without feeder layers.

ESCs can be detected by protein markers including transcription factorOct4, alkaline phosphatase (AP), stage-specific embryonic antigenSSEA-1, stage-specific embryonic antigen SSEA-3, stage-specificembryonic antigen SSEA-4, transcription factor NANOG, tumor rejectionantigen 1-60 (TRA-1-60), tumor rejection antigen 1-81 (TRA-1-81), SOX2,or REX1.

2. Induced Pluripotent Stem Cells

In other aspects, the precursor cells are derived from inducedpluripotent stem cells, commonly abbreviated iPS cells or iPSCs. Theinduction of pluripotency was originally achieved in 2006 using mousecells (Yamanaka et al. 2006) and in 2007 using human cells (Yu et al.2007; Takahashi et al. 2007) by reprogramming of somatic cells via theintroduction of transcription factors that are linked to pluripotency.The use of iPSCs circumvents most of the ethical and practical problemsassociated with large-scale clinical use of ES cells, and patients withiPSC-derived autologous transplants may not require lifelongimmunosuppressive treatments to prevent graft rejection.

With the exception of certain cell types (such as germ cells andenucleated erythrocytes), any cell can be used as a starting point foriPSCs. For example, cell types could be neurons, keratinocytes,fibroblasts, hematopoietic cells, mesenchymal cells, liver cells, orstomach cells. There is no limitation on the degree of celldifferentiation or the age of an animal from which cells are collected;even undifferentiated progenitor cells (including somatic stem cells)and finally differentiated mature cells can be used as sources ofsomatic cells in the methods disclosed herein. The somatic cell can bean adult or a fetal somatic cell. iPSCs can be grown under conditionsthat are known to differentiate human ESCs into specific cell types, andexpress human ESC markers including: SSEA-1, SSEA-3, SSEA-4, TRA-1-60,and TRA-1-81.

Somatic cells can be reprogrammed to produce induced pluripotent stemcells (iPSCs) using methods known to one of skill in the art. One ofskill in the art can readily produce induced pluripotent stem cells, seefor example, U.S. Patent Publication Nos. 20090246875, 201000210014;20120276636; U.S. Pat. Nos. 8,058,065, 8,129,187, 8,278,620, 8,268,630;and PCT Publication No. WO 2007069666, which are incorporated herein byreference. Generally, nuclear reprogramming factors are used to producepluripotent stem cells from a somatic cell. Reprogramming factors knownin the art include Klf4, c-Myc, Oct3/4, Sox2, Nanog, and Lin28. Anycombination of factors may be used in the present methods.

Mouse and human cDNA sequences of these nuclear reprogramming substancesare available with reference to the NCBI accession numbers mentioned inU.S. Pat. No. 8,183,038 and PCT Publication No. WO 2007069666, which areincorporated herein by reference. Methods for introducing one or morereprogramming substances, or nucleic acids encoding these reprogrammingsubstances, are known in the art, and disclosed for example, inpublished U.S. Pat. Nos. 8,071,369, 8,268,620, 8,691,574, 8,741,648,8,546,140, 8,900,871 and 9,175,268, which are incorporated herein byreference.

Once derived, iPSCs can be cultured in a medium sufficient to maintainpluripotency. The iPSCs may be used with various media and techniquesdeveloped to culture pluripotent stem cells, more specifically,embryonic stem cells, as described in U.S. Pat. No. 7,442,548 and U.S.Patent Publication. No. 20030211603. In the case of mouse cells, theculture may be carried out with the addition of Leukemia InhibitoryFactor (LIF) as a differentiation suppression factor to an ordinarymedium. In the case of human cells, basic fibroblast growth factor(bFGF) may be added in place of LIF. Other methods for the culture andmaintenance of iPSCs, as would be known to one of skill in the art, maybe used with the present disclosure.

In certain embodiments, undefined conditions may be used; for example,pluripotent cells may be cultured on fibroblast feeder cells or a mediumthat has been exposed to fibroblast feeder cells in order to maintainthe stem cells in an undifferentiated state. In some embodiments, thecell is cultured in the co-presence of mouse embryonic fibroblaststreated with radiation or an antibiotic to terminate the cell division,as feeder cells. Alternately, pluripotent cells may be cultured andmaintained in an essentially undifferentiated state using a defined,feeder-independent culture system, such as a TESR™ medium (Ludwig etal., 2006a; Ludwig et al., 2006b) or E8™/Essential 8™ medium (Chen etal., 2011).

Plasmids have been designed with a number of goals in mind, such asachieving regulated high copy number and avoiding potential causes ofplasmid instability in bacteria, and providing means for plasmidselection that are compatible with use in mammalian cells, includinghuman cells. Particular attention has been paid to the dual requirementsof plasmids for use in human cells. First, they are suitable formaintenance and fermentation in E. coli, so that large amounts of DNAcan be produced and purified. Second, they are safe and suitable for usein human patients and animals. The first requirement calls for high copynumber plasmids that can be selected for and stably maintainedrelatively easily during bacterial fermentation. The second requirementcalls for attention to elements such as selectable markers and othercoding sequences. In some embodiments, plasmids that encode a marker arecomposed of: (1) a high copy number replication origin, (2) a selectablemarker, such as, but not limited to, the neo gene for antibioticselection with kanamycin, (3) transcription termination sequences,including the tyrosinase enhancer and (4) a multicloning site forincorporation of various nucleic acid cassettes; and (5) a nucleic acidsequence encoding a marker operably linked to the tyrosinase promoter.There are numerous plasmid vectors that are known in the art forinducing a nucleic acid encoding a protein. These include, but are notlimited to, the vectors disclosed in U.S. Pat. Nos. 6,103,470;7,598,364; 7,989,425; and 6,416,998, which are incorporated herein byreference.

An episomal gene delivery system can be a plasmid, an Epstein-Barr virus(EBV)-based episomal vector (U.S. Pat. No. 8,546,140), a yeast-basedvector, an adenovirus-based vector, a simian virus 40 (SV40)-basedepisomal vector, a bovine papilloma virus (BPV)-based vector, or alentiviral vector. A viral gene delivery system can be an RNA-based orDNA-based viral vector (PCT/JP2009/062911, PCT/JP2011/069588;incorporated herein by reference).

3. Embryonic Stem Cells Derived by Somatic Cell Nuclear Transfer

Pluripotent stem cells for deriving the starting population of hostcells could also be prepared by means of somatic cell nuclear transfer,in which a donor nucleus is transferred into a spindle-free oocyte. Stemcells produced by nuclear transfer are genetically identical to thedonor nuclei. In one method, donor fibroblast nuclei from skinfibroblasts of a rhesus macaque are introduced into the cytoplasm ofspindle-free, mature metaphase II rhesus macaque ooctyes byelectrofusion (Byrne et al., 2007). The fused oocytes are activated byexposure to ionomycin, then incubated until the blastocyst stage. Theinner cell mass of selected blastocysts are then cultured to produceembryonic stem cell lines. The embryonic stem cell lines show normal EScell morphology, express various ES cell markers, and differentiate intomultiple cell types both in vitro and in vivo.

B. Precursor Cells

Certain embodiments of the present disclosure concern precursor cellscomprising a vector encoding a suicide gene under the control of acell-cycle dependent promoter. The precursor cells may be differentiatedfrom PSCs. Exemplary precursor cells include neural precursor cells(NPCs), cardiomyocyte precursor cells and pancreatic precursor cells.

Neural precursor cells may be differentiated from PSCs using methodsknown in the art such as, but not limited to, the method disclosed inU.S. Pat. No. 7,968,337; incorporated herein by reference. In brief, thePSCs are cultured in a first medium comprising basic fibroblast growthfactor (bFGF), a second medium comprising bFGF and epidermal growthfactor (EGF), and a third medium comprising bFGF and platelet-derivedgrowth factor (PDGF) to obtain neural precursor cells. The NPCs can bedetected and/or isolated using markers such as musashi, nestin, sox2,vimentin, pax6, and sox1. The NPCs can also be further differentiated toexpress a variety of neuronal markers, e.g., MAP2, beta-III-tubulin,synapsin, cholinacetyltransferase, tyrosin hydroxylase, GABA, glutamate,serotonin, peripherin and calbindin. Maturation and survival of thedifferentiated neurons can be enhanced by addition of neurotrophins,e.g., BDNF or neurotrophin 3 (NT-3). Additional methods for theproduction and culturing of NPCs can be found, for example, in U.S. Pat.Nos. 8,093,053; 5,980,885; 7,968,337 and 8,178,349 and U.S. ApplicationNo. 20100323444; each of which is incorporated herein by reference.

III. POLYNUCLEOTIDES OF THE PRESENT DISCLOSURE

In certain embodiments, the present disclosure concerns isolated andrecombinant polynucleotides, such as polynucleotides comprising a cellcycle-dependent promoter operatively linked to a suicide gene. Inparticular embodiments, the present disclosure concerns isolated nucleicacids and recombinant vectors incorporating nucleic acid sequences thatencode a suicide gene, the expression of which is operatively linked toa cell cycle-dependent promoter. The term “recombinant” may be used inconjunction with a polynucleotide or polypeptide and generally refers toa polypeptide or polynucleotide produced and/or manipulated in vitro orthat is a replication product of such a molecule.

A nucleic acid may be made by any technique known to one of ordinaryskill in the art. Non-limiting examples of a synthetic nucleic acid,particularly a synthetic oligonucleotide, include a nucleic acid made byin vitro chemical synthesis using phosphotriester, phosphite orphosphoramidite chemistry and solid phase techniques such as describedin EP 266,032, or via deoxynucleoside H-phosphonate intermediates asdescribed by Froehler et al., 1986, and U.S. Pat. No. 5,705,629. Anon-limiting example of enzymatically produced nucleic acid includes oneproduced by enzymes in amplification reactions such as PCR™ (see forexample, U.S. Pat. Nos. 4,683,202 and 4,682,195), or the synthesis ofoligonucleotides described in U.S. Pat. No. 5,645,897. A non-limitingexample of a biologically produced nucleic acid includes recombinantnucleic acid production in living cells, such as recombinant DNA vectorproduction in bacteria (see for example, Sambrook et al. 1989).

The nucleic acids used in the present disclosure can be combined withother nucleic acid sequences, such as promoters, polyadenylationsignals, additional restriction enzyme sites, multiple cloning sites,other coding segments, and the like, such that their overall length mayvary considerably. It is therefore contemplated that a nucleic acidfragment of almost any length may be employed, with the total lengthpreferably being limited by the ease of preparation and use in theintended recombinant nucleic acid protocol.

A. Nucleic Acid Delivery

The polynucleotides of the present disclosure may be introduced (e.g.,transfected or transduced) into a host cell by viral or non-viralmethods. Vectors provided herein are designed, primarily, to express asuicide gene under the control of a cell-cycle dependent promoter. Oneof skill in the art would be well-equipped to construct a vector throughstandard recombinant techniques (see, for example, Sambrook et al., 2001and Ausubel et al., 1996, both incorporated herein by reference).Vectors include but are not limited to, plasmids, cosmids, viruses(e.g., bacteriophage, animal viruses, and plant viruses), artificialchromosomes (e.g., YACs), retroviral vectors (e.g. derived from Moloneymurine leukemia virus vectors (MoMLV), MSCV, SFFV, MPSV, SNV etc),lentiviral vectors (e.g. derived from HIV-1, HIV-2, SIV, BIV, FIV etc.),adenoviral (Ad) vectors including replication competent, replicationdeficient and gutless forms thereof, adeno-associated viral (AAV)vectors, simian virus 40 (SV-40) vectors, bovine papilloma virusvectors, Epstein-Barr virus vectors, herpes virus vectors, vacciniavirus vectors, Harvey murine sarcoma virus vectors, murine mammary tumorvirus vectors, Rous sarcoma virus vectors, parvovirus vectors, poliovirus vectors, vesicular stomatitis virus vectors, maraba virus vectorsand group B adenovirus enadenotucirev vectors.

1. Viral Vectors

Viral vectors encoding a cell cycle-dependent promoter operativelylinked to a suicide gene may be provided in certain aspects of thepresent disclosure. In generating recombinant viral vectors,non-essential genes are typically replaced with a gene or codingsequence for a heterologous (or non-native) protein. A viral vector is akind of expression construct that utilizes viral sequences to introducenucleic acid and possibly proteins into a cell. The ability of certainviruses to infect cells or enter cells via receptor-mediatedendocytosis, and to integrate into host cell genomes and express viralgenes stably and efficiently have made them attractive candidates forthe transfer of foreign nucleic acids into cells (e.g., mammaliancells). Non-limiting examples of virus vectors that may be used todeliver a nucleic acid of certain aspects of the present disclosure aredescribed below. “Recombinant viral vectors” in the present disclosurerefers to viral vectors constructed by genetic recombination techniques.Viral vectors constructed using packaging cells and DNAs encoding aviral genome are called recombinant viral vectors.

i. Retroviral Vectors

In one aspect of the present disclosure, retroviral constructs areprovided comprising a 5′ long terminal repeat (LTR), a tRNA bindingsite, a packaging signal, one or more heterologous sequences, an originof second strand DNA synthesis and a 3′ LTR, wherein the vectorconstruct lacks gag/pol and/or env coding sequences.

Heterologous sequences that are included in the vector construct arethose that encode a protein, preferably a suicide gene, such as herpessimplex virus thymidine kinase. Within certain embodiments of thepresent disclosure, the expression cassette described herein may becontained within a plasmid construct.

A retroviral vector of the present disclosure includes at least oneexpression cassette, which is an assembly that is capable of directingthe expression of the sequences(s) or gene(s) of interest. Theexpression cassette includes a transcriptional promoter region orpromoter/enhancer that is operatively linked to the sequence(s) orgene(s) of interest, and may include a polyadenylation sequence as well.Such vector constructs also include a packaging signal, LTRs orfunctional portions thereof, and positive and negative strand primerbinding sites appropriate to the retrovirus used. Optionally, therecombinant retroviral vector may also include a selectable and/ornon-selectable marker, an origin of second strand DNA synthesis, asignal that allows the plasmid construct to exist as single-stranded DNA(e.g., a M13 origin of replication), a bacterial origin of replication,and a mammalian origin of replication (e.g., a SV40 or adenovirus originof replication), one or more restriction sites, and a translationtermination sequence. Examples of selectable and non-selectable markersinclude, but are not limited to, neomycin (Neo), thymidine kinase (TK),hygromycin, phleomycin, puromycin, histidinol, green fluorescent protein(GFP), human placental alkaline phosphatase (PLAP), DHFR,β-galactosidase, and human growth hormone (hGH).

In retroviruses, the LTR may also be modified. The LTR is aretrovirus-specific sequence, which is present at both ends of the viralgenome. The 5′ LTR serves as a promoter, enhancing proviral mRNAtranscription. Thus, it may be possible to enhance mRNA transcription ofthe gene transfer vector, improve packaging efficiency, and increasevector titer if the portion exhibiting the 5′ LTR promoter activity inthe gene transfer vector is substituted with another promoter havingstronger promoter activity. Furthermore, for example, in the case oflentiviruses, the viral protein tat is known to enhance 5′ LTRtranscription activity, and therefore, substitution of the 5′ LTR with apromoter independent of the tat protein will enable the exclusion of tatfrom the packaging vectors. After RNAs of viruses that have infected orinvaded cells are reverse transcribed, the LTRs at both ends are linkedto form a closed circular structure, viral integrase couples with thelinkage site, and this structure is then integrated into cellchromosomes. The transcribed proviral mRNAs consist of the regionranging from the 5′ LTR transcription initiation site to the 3′ LTRpolyadenylation sequence located downstream. The 5′ LTR promoter portionis not packaged in the virus. Thus, even if the promoter is replacedwith another sequence, the portion integrated into target cellchromosomes is unchanged. Based on the facts described above,substitution of the 5′ LTR promoter is thought to provide a safer vectorwith a higher titer. Thus, substitution of the promoter at the 5′ end ofa gene transfer vector can increase the titer of a packageable vector.

Safety can be improved in recombinant retroviral virus vectors bypreventing transcription of the full-length vector mRNA in target cells.This is achieved using a self-inactivating vector (SIN vector) preparedby partially eliminating the 3′ LTR sequence. The provirus that hasinvaded the target cell chromosomes has its 5′ end bound to the U3portion of its 3′ LTR. Thus, the U3 portion is located at the 5′ end inthe gene transfer vector, and from that point, the entire RNA of thegene transfer vector is transcribed. If there are retroviruses orsimilar proteins in target cells, it is possible that the gene transfervector may be re-packaged and infect other cells. There is also apossibility that the 3′ LTR promoter may express host genes locateddownstream of the viral genome. When the 3′ LTR U3 portion is deletedfrom a gene transfer vector, target cells lack the promoters of 5′ LTRand 3′ LTR, thereby preventing the transcription of the full-lengthviral RNA and host genes. Furthermore, since only the genes of interestare transcribed from endogenous promoters, highly safe vectors capableof high expression can be expected. Such vectors are preferable in thepresent disclosure. SIN vectors can be constructed according to knownmethods.

SIN vectors have the added advantage of overcoming the gradual decreasein expression of introduced genes resulting from host methylation of LTRsequences after integration (Challita, P. M. and Kohn, D. B., Proc.Natl. Acad. Sci. USA 91: 2567, 1994). LTR methylation hardly reducesgene expression level in SIN vectors. This is because the vector losesmost of the LTR sequence upon integration into the host genome. A SINvector prepared by substituting another promoter sequence for the 3′ LTRU3 region of a gene transfer vector was found to maintain a stableexpression for more than two months after introduction into primate EScells (WO 02/101057). Thus, a SIN vector designed to self-inactivate bythe modification of the LTR U3 region may be used in the presentdisclosure.

Retroviruses can be produced by transcribing in host cells gene transfervector DNAs which contain a packaging signal and forming virus particlesin the presence of gag, pol and envelope proteins. The packaging signalsequence encoded by the gene transfer vector DNAs should preferably besufficient in length to maintain the structure formed by the sequence.However, in order to suppress the frequency of wild-type virusformation, which occurs due to recombination of the vector DNA packagingsignal and the packaging vector supplying the gag and pol proteins, itis also necessary to keep sequence overlapping between these vectorsequences to a minimum. Therefore, when it comes to the construction ofthe gene transfer vector DNAs, it is preferable to use a sequence whichis as short as possible and yet still contains the sequence essentialfor packaging, to ensure packaging efficiency and safety.

The SIV vectors may be replication-incompetent viruses from which 40% ormore, more preferably 50% or more, still more preferably 60% or more,even more preferably 70% or more, and most preferably 80% or more of thesequence derived from the original SIV genome has been removed.

In a gene transfer vector DNA, the gag protein has been modified suchthat it is not expressed. Viral gag protein may be detected by a livingbody as a foreign substance, and thus as a potential antigen.Alternatively, the protein may affect cellular functions. To prevent gagprotein expression, nucleotides downstream of the gag start codon can beadded or deleted, introducing modifications which will cause aframeshift. It is also preferable to delete portions of the codingregion of the gag protein. The 5′ portion of the coding region of thegag protein is known to be essential for virus packaging. Thus, in agene transfer vector, it is preferable that the C-terminal side of thegag protein-coding region has been deleted. It is preferable to deleteas large a portion of the gag coding region as possible, so long as thedeletion does not considerably affect the packaging efficiency. It isalso preferable to replace the start codon (ATG) of the gag protein witha codon other than ATG. The replacement codon can be selectedappropriately so as not to greatly affect the packaging efficiency. Aviral vector can be produced by introducing the constructed genetransfer vector DNA, which comprises the packaging signal, intoappropriate packaging cells. The viral vector produced can be recoveredfrom, for example, the culture supernatant of packaging cells.

“Packaging cell” refers to a cell that contains those elements necessaryfor production of infectious recombinant retrovirus that are lacking ina recombinant retroviral vector. Packaging cells contain one or moreexpression cassettes that are capable of expressing proteins that encodegag, pol, and env-derived proteins. Packaging cells can also containexpression cassettes encoding one or more of vif, rev, or ORF 2 inaddition to gag/pol and env expression cassettes.

There is no limitation on the type of packaging cell, as long as thecell line is generally used in viral production. When used for humangene therapy, a human- or monkey-derived cell is suitable. Human celllines that can be used as packaging cells include, for example, 293cells, 293T cells, 293EBNA cells, SW480 cells, u87MG cells, HOS cells,C8166 cells, MT-4 cells, Molt-4 cells, HeLa cells, HT1080 cells, andTE671 cells. Monkey cell lines include, for example, COS1 cells, COSTcells, CV-1 cells, and BMT10 cells.

Lentiviruses are complex retroviruses, which, in addition to the commonretroviral genes gag, pol, and env, contain other genes with regulatoryor structural function. Lentiviral vectors are well known in the art(see, for example, Naldini et al., 1996; Zufferey et al., 1997; Blomeret al., 1997; U.S. Pat. Nos. 6,013,516 and 5,994,136). Recombinantlentiviral vectors are capable of infecting non-dividing cells and canbe used for both in vivo and ex vivo gene transfer and expression ofnucleic acid sequences. For example, recombinant lentivirus capable ofinfecting a non-dividing cell—wherein a suitable host cell istransfected with two or more vectors carrying the packaging functions,namely gag, pol and env, as well as rev and tat—is described in U.S.Pat. No. 5,994,136, incorporated herein by reference.

The pseudotyped lentiviral vectors of the present disclosure can bepurified to become substantially pure. The phrase “substantially pure”means that the pseudotyped lentiviral vectors contain substantially noreplicable virus other than the lentivirus. The purification can beachieved using known purification and separation methods such asfiltration, centrifugation, and column purification. For example, avector can be precipitated and concentrated by filtering a vectorsolution with a 0.45-μm filter, and then centrifuging it at 42500×g at4° C. for 90 minutes. If necessary, the pseudotyped lentiviral vectorsof the present disclosure can be prepared as compositions byappropriately combining with desired pharmaceutically acceptablecarriers or vehicle. Specifically, the vector can be appropriatelycombined with, for example, sterilized water, physiological saline,culture medium, serum, and phosphate buffered saline (PBS). The vectorcan also be combined with a stabilizer, biocide, and such. Compositionscontaining a pseudotyped lentiviral vector of the present disclosure areuseful as reagents or pharmaceuticals. For example; compositions of thepresent disclosure can be used as reagents for gene transfer into airwaystem cells, or as pharmaceuticals for gene therapy of various diseasessuch as genetic diseases.

Within one aspect of the present disclosure, retroviral gene deliveryvehicles are provided that are constructed to carry or express aselected gene(s) or sequence(s) of interest. Briefly, retroviral genedelivery vehicles of the present disclosure may be readily constructedfrom a wide variety of retroviruses, including, for example, B, C, and Dtype retroviruses as well as spumaviruses and lentiviruses, such as FIV,HIV-1, HIV-2, EIAV, and SIV (see RNA Tumor Viruses, Second Edition, ColdSpring Harbor Laboratory, 1985). Such retroviruses may be readilyobtained from depositories or collections, such as the American TypeCulture Collection (“ATCC”; 10801 University Blvd., Manassas, Va.20110-2209), or isolated from known sources using commonly availabletechniques. Any of the above retroviruses may be readily utilized inorder to assemble or construct retroviral gene delivery vehicles giventhe disclosure provided herein, and standard recombinant techniques(e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, 2d ed.,Cold Spring Harbor Laboratory Press, 1989; Kunkle, PNAS 52:488, 1985).In addition, within certain embodiments of the present disclosure,portions of the retroviral gene delivery vehicles may be derived fromdifferent retroviruses. For example, within one embodiment of thepresent disclosure, retroviral LTRs may be derived from a murine sarcomavirus, a tRNA binding site from a Rous sarcoma virus, a packaging signalfrom a murine leukemia virus, and an origin of second strand synthesisfrom an avian leukosis virus.

Within certain embodiments of the present disclosure, retroviral vectorsare provided wherein viral promoters, preferably CMV or SV40 promotersand/or enhancers are utilized to drive expression of one or more genesof interest. Within other aspects of the present disclosure, retroviralvectors are provided wherein tissue-specific promoters are utilized todrive expression of one or more genes of interest.

Retrovirus vector constructs for use with the present disclosure may begenerated such that more than one gene of interest is expressed andpreferably secreted. This may be accomplished through the use of di- oroligo-cistronic cassettes (e.g., where the coding regions are separatedby 120 nucleotides or less, see generally Levin et al., Gene108:167-174, 1991), or through the use of internal ribosome entry sites(“IRES”).

Within one aspect of the present disclosure, self-inactivating (SIN)vectors are made by deleting promoter and enhancer elements in the U3region of the 3′ LTR, including the TATA box, and binding sites for oneor more transcription factors. The deletion is transferred to the 5′ LTRafter reverse transcription and integration in transduced cells. Thisresults in the transcriptional inactivation of the LTR in the provirus.Possible advantages of SIN vectors include increased safety of the genedelivery system as well as the potential to reduce promoter interferencebetween the LTR and the internal promoter, which may result in increasedexpression of the gene of interest. Furthermore, it is reasonable toexpect tighter control of inducible gene therapy vectors due to the lackof an upstream promoter element in the 5′ LTR.

Within one aspect of the present disclosure, lentiviral vectorconstructs are provided comprising a 5′ LTR, a tRNA binding site, apackaging signal, one or more heterologous sequences, an origin ofsecond strand DNA synthesis, an RNA export element, and a 3′ LTR.Briefly, long terminal repeats (“LTRs”) are subdivided into threeelements, designated U5, R, and U3. These elements contain a variety ofsignals that are responsible for the biological activity of aretrovirus, including for example, promoter and enhancer elements thatare located within U3. LTRs may be readily identified in the provirus(integrated DNA form) due to their precise duplication at either end ofthe genome. For purposes of the present disclosure, a 5′ LTR should beunderstood to include as much of the native 5′ LTR as is required tofunction as a 5′ promoter or promoter/enhancer element, to allow reversetranscription, and to allow integration of the DNA form of the vector.The 3′ LTR should be understood to include as much of the 3′ FIV LTR asis required to function as a polyadenylation signal, to allow reversetranscription, and to allow integration of the DNA form of the vector.

Additionally, retroviral vectors may contain hybrid LTRs where up to 75%of the wild-type LTR sequence is deleted and replaced by one or moreviral or non-viral promoter or promoter/enhancer elements (e.g., otherretroviral LTRs and/or non-retroviral promoters or promoter/enhancerssuch as the CMV promoter/enhancer or the SV40 promoter) similar to thehybrid LTRs described by Chang, et al., J. Virology 67, 743-752, 1993;Finer, et al., Blood 83, 43-50, 1994 and Robinson, et al., Gene Therapy2, 269-278, 1995.

The tRNA binding site and origin of second strand DNA synthesis are alsoimportant for a retrovirus to be biologically active, and may be readilyidentified by one of skill in the art. For example, tRNA binds to aretroviral tRNA binding site by Watson-Crick base pairing, and iscarried with the retrovirus genome into a viral particle. The tRNA isthen utilized as a primer for DNA synthesis by reverse transcriptase.The tRNA binding site may be readily identified based upon its locationjust downstream from the 5′ LTR. Similarly, the origin of second strandDNA synthesis is, as its name implies, important for the second strandDNA synthesis of a retrovirus. This region, which is also referred to asthe poly-purine tract, is located just upstream of the 3′ LTR.

In addition to 5′ and 3′ LTRs, a tRNA binding site, a packaging signal,and an origin of second strand DNA synthesis, certain preferredrecombinant retroviral vector constructs for use herein also compriseone or more genes of interest. In addition, the retroviral vectors may,but need not, include an RNA export element (also variously referred toas RNA transport, nuclear transport or nuclear export elements) that maybe a RRE (Rev-responsive element) or a heterologous transport element.Representative examples of suitable heterologous RNA export elementsinclude the Mason-Pfizer monkey virus constitutive transport element(Bray et al., PNAS USA 91, 1256-1260, 1994), the hepatitis B virusposttranscriptional regulatory element (Huang et al., Mol Cell. Biol.73:7476-7486, 1993 and Huang et al., J. Virology 65:3193-3199, 1994), orlentiviral Rev-responsive elements (Daly et al., Nature 342:816-819,1989 and Zapp et al., Nature 342:714-716, 1989).

Retroviral vector constructs that lack both gag/pol and env codingsequences may be used with the present disclosure. As utilized herein,the phrase “lacks gag/pol or env coding sequences” should be understoodto mean that the vector contains less than 20, preferably less than 15,more preferably less than 10, and most preferably less than 8consecutive nucleotides that are found in gag/pol or env genes, and inparticular, within gag/pol or env expression cassettes that are used toconstruct packaging cell lines for the retroviral vector construct. Thisaspect of the present disclosure provides for retroviral vectors havinga low probability of undesirable recombination with gag/pol or envsequences that may occur in a host cell or be introduced therein, forexample, by transformation with an expression cassette. The productionof retroviral vector constructs lacking gag/pol or env sequences may beaccomplished by partially eliminating the packaging signal and/or theuse of a modified or heterologous packaging signal. Within otherembodiments of the present disclosure, retroviral vector constructs areprovided wherein a portion of the packaging signal that may extend into,or overlap with, the retroviral gag/pol sequence is modified (e.g.,deleted, truncated, or bases exchanged). Within other aspects of thepresent disclosure, retroviral vector constructs are provided thatinclude the packaging signal that may extend beyond the start of thegag/pol gene. Within certain embodiments, the packaging signal that mayextend beyond the start of the gag/pol gene is modified in order tocontain one, two, or more stop codons within the gag/pol reading frame.Most preferably, one of the stop codons eliminates the gag/pol startsite. In other embodiments, the introduced mutation may cause a frameshift in the gag/pol coding region.

Other retroviral gene delivery vehicles may likewise be utilized withinthe context of the present disclosure, including for example thosedescribed in EP 0,415,731; WO 90/07936; WO 91/0285, WO 9403622; WO9325698; WO 9325234; U.S. Pat. No. 5,219,740; WO 9311230; WO 9310218;Vile and Hart, Cancer Res. 53:3860-3864, 1993; Vile and Hart, CancerRes. 53:962-967, 1993; Ram et al., Cancer Res. 53:83-88, 1993; Takamiyaet al., J. Neurosci. Res. 33:493-503, 1992; Baba et al., J. Neurosurg.79:729-735, 1993 (U.S. Pat. No. 4,777,127, GB 2,200,651, EP 0,345,242and WO91/02805).

Packaging cell lines suitable for use with the above describedretroviral constructs may be readily prepared (see, e.g., U.S. Pat. Nos.5,591,624 and 6,013,517; and International Publication No. WO 95/30763),and utilized to create producer cell lines for the production ofrecombinant vector particles. The parent cell line from which thepackaging cell line is derived can be selected from a wide variety ofmammalian cell lines, including for example, human cells, monkey cells,feline cells, dog cells, mouse cells, and the like.

After selection of a suitable host cell for the generation of apackaging cell line, one or more expression cassettes are introducedinto the cell line in order to complement or supply in trans componentsof the vector which have been deleted (see, e.g., U.S. Pat. Nos.5,591,624 and 6,013,517, incorporated herein by reference in theirentireties; and International Publication No. WO 95/30763). For example,packaging expression cassettes may encode either gag/pol sequencesalone, gag/pol sequences and one or more of vif, rev, or ORF 2, or oneor more of vif, rev, or ORF 2 alone and may contain an RNA exportelement. For example, the packaging cell line may contain only ORF 2,vif, or rev alone, ORF 2 and vif, ORF 2 and rev, vif and rev, or allthree of ORF 2, vif and, rev.

Packaging cell lines may also comprise a promoter and a sequenceencoding ORF 2, vif, rev, or an envelope (e.g., VSV-G), wherein thepromoter is operatively linked to the sequence encoding ORF 2, vif, rev,or the envelope. For packaging cell lines containing inducible gag/polor env expression cassettes, additional expression cassettesfacilitating the transactivation of the inducible promoter may beincorporated. The expression cassette may or may not be stablyintegrated. The packaging cell line, upon introduction of a retroviralvector, may produce particles at a concentration of greater than 103,104, 105,106, 107, 108, or 109 cfu/mL.

ii. Lentiviral Vectors

“Lentivirus” refers to a virus belonging to the lentivirus genus.Lentiviruses include, but are not limited to, human immunodeficiencyvirus (HIV) (for example, HIV-1 or HIV-2), simian immunodeficiency virus(SIV), feline immunodeficiency virus (FIV), Maedi-Visna-like virus(EV1), equine infectious anemia virus (EIAV) and caprine arthritisencephalitis virus (CAEV).

The terms “lentiviral vector construct,” “lentiviral vector,” and“recombinant lentiviral vector” are used interchangeably herein andrefer to a nucleic acid construct derived from a lentivirus that carriesand, within certain embodiments, is capable of directing the expressionof a nucleic acid molecule of interest. Lentiviral vectors can have oneor more of the lentiviral wild-type genes deleted in whole or part butretain functional flanking long-terminal repeat (LTR) sequences. TheLTRs need not be the wild-type nucleotide sequences, and may be altered,e.g., by the insertion, deletion or substitution of nucleotides, so longas the sequences provide for functional rescue, replication, andpackaging. The lentiviral vector may also contain a selectable marker.

The term “recombinant lentivirus” refers to a virus particle thatcontains a lentivirus-derived viral genome, lacks the self-renewalability, and has the ability to introduce a nucleic acid molecule into ahost. For example, the recombinant lentiviruses of the presentdisclosure include virus particles comprising a nucleic acid moleculethat comprises a lentiviral genome-derived packaging signal sequence.The recombinant lentivirus is capable of reverse transcribing itsgenetic material into DNA and incorporating this genetic material into ahost cell's DNA upon infection. Recombinant lentivirus particles mayhave a lentiviral envelope, a non-lentiviral envelope (e.g., anamphotropic or VSV-G envelope), a chimeric envelope, or a modifiedenvelope (e.g., truncated envelopes or envelopes containing hybridsequences).

A nucleic acid carried by a lentiviral vector of the present disclosurecan be introduced into pluripotent stem cells or neural progenitor cellsby contacting this vector with pluripotent stem cells of primates,including humans, or rodents, including mice and rats. The presentdisclosure relates to methods for introducing suicide genes intopluripotent stem cells, which comprise the step of contactingpluripotent stem cells with the vectors of the present disclosure. Thepluripotent stem cells targeted for gene introduction are notparticularly limited and, for example, include embryonic stem cells orinduced pluripotent stem cells.

iii. Adenoviral Vector

The polynucleotide encoding a cell-cycle dependent promoter operativelylinked to a suicide gene may be provided in an adenoviral vector.Although adenovirus vectors are known to have a low capacity forintegration into genomic DNA, this feature is counterbalanced by thehigh efficiency of gene transfer afforded by these vectors. Adenovirusexpression vectors include constructs containing adenovirus sequencessufficient to (a) support packaging of the construct and (b) toultimately express a recombinant gene construct that has been clonedtherein.

Adenovirus growth and manipulation is known to those of skill in theart, and exhibits broad host range in vitro and in vivo. This group ofviruses can be obtained in high titers, e.g., 109-1011 plaque-formingunits per ml, and they are highly infective. The life cycle ofadenovirus does not require integration into the host cell genome. Theforeign genes delivered by adenovirus vectors are episomal and,therefore, have low genotoxicity to host cells. No side effects havebeen reported in studies of vaccination with wild-type adenovirus (Couchet al., 1963; Top et al., 1971), demonstrating their safety andtherapeutic potential as in vivo gene transfer vectors.

Knowledge of the genetic organization of adenovirus, a 36 kb, linear,double-stranded DNA virus, allows substitution of large pieces ofadenoviral DNA with foreign sequences up to 7 kb (Grunhaus and Horwitz,1992). In contrast to retrovirus, the adenoviral infection of host cellsdoes not result in chromosomal integration because adenoviral DNA canreplicate in an episomal manner without potential genotoxicity. Also,adenoviruses are structurally stable, and no genome rearrangement hasbeen detected after extensive amplification.

Adenovirus may be used as a gene transfer vector because of itsmid-sized genome, ease of manipulation, high titer, wide target-cellrange and high infectivity. Both ends of the viral genome contain100-200 base pair inverted repeats (ITRs), which are cis elementsnecessary for viral DNA replication and packaging. The early (E) andlate (L) regions of the genome contain different transcription unitsthat are divided by the onset of viral DNA replication. The E1 region(E1A and E1B) encodes proteins responsible for the regulation oftranscription of the viral genome and a few cellular genes. Theexpression of the E2 region (E2A and E2B) results in the synthesis ofthe proteins for viral DNA replication. These proteins are involved inDNA replication, late gene expression and host cell shut-off (Renan,1990). The products of the late genes, including the majority of theviral capsid proteins, are expressed only after significant processingof a single primary transcript issued by the major late promoter (MLP).The MLP, (located at 16.8 m.u.) is particularly efficient during thelate phase of infection, and all the mRNAs issued from this promoterpossess a 5′-tripartite leader (TPL) sequence which makes themparticular mRNA's for translation.

A recombinant adenovirus provided herein can be generated fromhomologous recombination between a shuttle vector and provirus vector.Due to the possible recombination between two proviral vectors,wild-type adenovirus may be generated from this process. Therefore, asingle clone of virus is isolated from an individual plaque and itsgenomic structure is examined.

The adenovirus vector may be replication defective, or at leastconditionally defective, the nature of the adenovirus vector is notbelieved to be crucial to the successful practice of the presentdisclosure. The adenovirus may be of any of the 42 different knownserotypes or subgroups A-F. Adenovirus type 5 of subgroup C is theparticular starting material in order to obtain the conditionalreplication-defective adenovirus vector for use in the presentdisclosure. This is because Adenovirus type 5 is a human adenovirusabout which a great deal of biochemical and genetic information isknown, and it has historically been used for most constructionsemploying adenovirus as a vector.

Nucleic acids can be introduced to adenoviral vectors as a position fromwhich a coding sequence has been removed. For example, a replicationdefective adenoviral vector can have the E1-coding sequences removed.The polynucleotide encoding the gene of interest may also be inserted inlieu of the deleted E3 region in E3 replacement vectors as described byKarlsson et al. (1986) or in the E4 region where a helper cell line orhelper virus complements the E4 defect.

Generation and propagation of replication deficient adenovirus vectorscan be performed with helper cell lines. One unique helper cell line,designated 293, was transformed from human embryonic kidney cells by Ad5DNA fragments and constitutively expresses E1 proteins (Graham et al.,1977). Since the E3 region is dispensable from the adenovirus genome(Jones and Shenk, 1978), adenovirus vectors, with the help of 293 cells,carry foreign DNA in either the E1, the E3, or both regions (Graham andPrevec, 1991).

Helper cell lines may be derived from human cells such as humanembryonic kidney cells, muscle cells, hematopoietic cells or other humanembryonic mesenchymal or epithelial cells. Alternatively, the helpercells may be derived from the cells of other mammalian species that arepermissive for human adenovirus. Such cells include, e.g., Vero cells orother monkey embryonic mesenchymal or epithelial cells. As stated above,a particular helper cell line is 293.

Methods for producing recombinant adenovirus are known in the art, suchas U.S. Pat. No. 6,740,320, incorporated herein by reference. Also,Racher et al. (1995) have disclosed improved methods for culturing 293cells and propagating adenovirus. In one format, natural cell aggregatesare grown by inoculating individual cells into 1 liter siliconizedspinner flasks (Techne, Cambridge, UK) containing 100-200 ml of medium.Following stirring at 40 rpm, the cell viability is estimated withtrypan blue. In another format, Fibra-Cel microcarriers (Bibby Sterlin,Stone, UK) (5 g/l) are employed as follows. A cell inoculum, resuspendedin 5 ml of medium, is added to the carrier (50 ml) in a 250 mlErlenmeyer flask and left stationary, with occasional agitation, for 1to 4 hours. The medium is then replaced with 50 ml of fresh medium andshaking initiated. For virus production, cells are allowed to grow toabout 80% confluence, after which time the medium is replaced (to 25% ofthe final volume) and adenovirus added at an MOI of 0.05. Cultures areleft stationary overnight, following which the volume is increased to100% and shaking commenced for another 72 hours.

iv. Adeno-associated Viral Vector

Adeno-associated virus (AAV) is may be used in the present disclosure asit has a high frequency of integration and it can infect nondividingcells, thus making it useful for delivery of genes into mammalian cells(Muzyczka, 1992). AAV has a broad host range for infectivity (Tratschin,et al., 1984; Laughlin, et al., 1986; Lebkowski, et al., 1988;McLaughlin, et al., 1988), which means it is applicable for use with thepresent disclosure. Details concerning the generation and use of rAAVvectors are described in U.S. Pat. Nos. 5,139,941 and 4,797,368.

AAV is a dependent parvovirus in that it requires coinfection withanother virus (e.g., adenovirus or a member of the herpes virus family)to undergo a productive infection in cultured cells (Muzyczka, 1992). Inthe absence of coinfection with helper virus, the wild-type AAV genomeintegrates through its ends into human chromosome 19 where it resides ina latent state as a provirus (Kotin et al., 1990; Samulski et al.,1991). rAAV, however, is not restricted to chromosome 19 for integrationunless the AAV Rep protein is also expressed (Shelling and Smith, 1994).When a cell carrying an AAV provirus is superinfected with a helpervirus, the AAV genome is rescued from the chromosome or from arecombinant plasmid, and a normal productive infection is established(Samulski et al., 1989; McLaughlin et al., 1988; Kotin et al., 1990;Muzyczka, 1992).

A recombinant AAV (rAAV) virus may be made by cotransfecting a plasmidcontaining the gene of interest flanked by the two AAV terminal repeats(McLaughlin et al., 1988; Samulski et al., 1989; each incorporatedherein by reference) and an expression plasmid containing the wild-typeAAV coding sequences without the terminal repeats, for example pIM45(McCarty et al., 1991). The cells can also be infected or transfectedwith adenovirus or plasmids carrying the adenovirus genes required forAAV helper function. rAAV virus stocks made in such fashion arecontaminated with adenovirus which must be physically separated from therAAV particles (for example, by cesium chloride density centrifugation).Alternatively, adenovirus vectors containing the AAV coding regions orcell lines containing the AAV coding regions and some or all of theadenovirus helper genes could be used (Yang et al., 1994; Clark et al.,1995). Cell lines carrying the rAAV DNA as an integrated provirus canalso be used (Flotte et al., 1995).

v. Other Viral Vectors

Other viral vectors may be employed as constructs in the presentdisclosure. Vectors derived from viruses such as vaccinia virus(Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988) andherpes simplex viruses may be employed. They offer several attractivefeatures for various mammalian cells (Friedmann, 1989; Ridgeway, 1988;Baichwal and Sugden, 1986; Coupar et al., 1988; Horwich et al., 1990).In some embodiments, the polynucleotide may be delivered usingintegrating adenovectors.

A molecularly cloned strain of Venezuelan equine encephalitis (VEE)virus has been genetically refined as a replication competent vaccinevector for the expression of heterologous viral proteins (Davis et al.,1996). Studies have demonstrated that VEE infection stimulates potentCTL responses and has been suggested that VEE may be an extremely usefulvector for immunizations (Caley et al., 1997).

In further embodiments, the polynucleotide is housed within an infectivevirus that has been engineered to express a specific binding ligand. Thevirus particle will thus bind specifically to the cognate receptors ofthe target cell and deliver the contents to the cell. Specific targetingof retrovirus vectors may be based on the chemical modification of aretrovirus by the chemical addition of lactose residues to the viralenvelope. This modification can permit the specific infection ofhepatocytes via sialoglycoprotein receptors.

For example, targeting of recombinant retroviruses was designed in whichbiotinylated antibodies against a retroviral envelope protein andagainst a specific cell receptor were used. The antibodies were coupledvia the biotin components by using streptavidin (Roux et al., 1989).Using antibodies against major histocompatibility complex class I andclass II antigens, they demonstrated the infection of a variety of humancells that bore those surface antigens with an ecotropic virus in vitro(Roux et al., 1989).

2. Other Methods of Nucleic Acid Delivery

In addition to viral delivery of the polynucleotides comprising a cellcycle-dependent promoter operatively linked to a suicide gene, thefollowing are additional methods of recombinant gene delivery to a givenhost cell and are thus considered in the present disclosure.

Introduction of a nucleic acid, such as DNA or RNA, may use any suitablemethods for nucleic acid delivery for transformation of a cell, asdescribed herein or as would be known to one of ordinary skill in theart. Such methods include, but are not limited to, direct delivery ofDNA such as by injection of naked DNA, nanoparticles, such as lipidnanoparticles, gene gun, ex vivo transfection (Wilson et al., 1989,Nabel et al, 1989), by injection (U.S. Pat. Nos. 5,994,624, 5,981,274,5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466 and5,580,859, each incorporated herein by reference), includingmicroinjection (Harland and Weintraub, 1985; U.S. Pat. No. 5,789,215,incorporated herein by reference); by electroporation (U.S. Pat. No.5,384,253, incorporated herein by reference; Tur-Kaspa et al., 1986;Potter et al., 1984); by calcium phosphate precipitation (Graham and VanDer Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990); by usingDEAE-dextran followed by polyethylene glycol (Gopal, 1985); by directsonic loading (Fechheimer et al., 1987); by liposome mediatedtransfection (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau etal., 1987; Wong et al., 1980; Kaneda et al., 1989; Kato et al., 1991)and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu, 1988);by microprojectile bombardment (PCT Application Nos. WO 94/09699 and95/06128; U.S. Pat. Nos. 5,610,042; 5,322,783 5,563,055, 5,550,318,5,538,877 and 5,538,880, and each incorporated herein by reference); byagitation with silicon carbide fibers (Kaeppler et al., 1990; U.S. Pat.Nos. 5,302,523 and 5,464,765, each incorporated herein by reference); byAgrobacterium-mediated transformation (U.S. Pat. Nos. 5,591,616 and5,563,055, each incorporated herein by reference); bydesiccation/inhibition-mediated DNA uptake (Potrykus et al., 1985), andany combination of such methods. Through the application of techniquessuch as these, organelle(s), cell(s), tissue(s) or organism(s) may bestably or transiently transformed.

i. Electroporation

In certain particular embodiments of the present disclosure, the geneconstruct is introduced into target cells via electroporation.Electroporation involves the exposure of cells (or tissues) and DNA (ora DNA complex) to a high-voltage electric discharge.

It is contemplated that electroporation conditions forhyperproliferative cells from different sources may be optimized. Onemay particularly wish to optimize such parameters as the voltage, thecapacitance, the time and the electroporation media composition. Theexecution of other routine adjustments will be known to those of skillin the art. See e.g., Hoffman, 1999; Heller et al., 1996.

ii. Lipid-Mediated Transformation

In a further embodiment, the polynucleotide comprising a cellcycle-dependent promoter operatively linked to a suicide gene may beentrapped in a liposome or lipid formulation. Liposomes are vesicularstructures characterized by a phospholipid bilayer membrane and an inneraqueous medium. Multilamellar liposomes have multiple lipid layersseparated by aqueous medium. They form spontaneously when phospholipidsare suspended in an excess of aqueous solution. The lipid componentsundergo self-rearrangement before the formation of closed structures andentrap water and dissolved solutes between the lipid bilayers (Ghosh andBachhawat, 1991). Also contemplated is a gene construct complexed withLipofectamine (Gibco BRL).

Lipid-mediated nucleic acid delivery and expression of foreign DNA invitro has been very successful (Nicolau and Sene, 1982; Fraley et al.,1979; Nicolau et al., 1987). Wong et al. (1980) demonstrated thefeasibility of lipid-mediated delivery and expression of foreign DNA incultured chick embryo, HeLa and hepatoma cells.

Lipid based non-viral formulations provide an alternative to adenoviralgene therapies. Although many cell culture studies have documented lipidbased non-viral gene transfer, systemic gene delivery via lipid basedformulations has been limited. A major limitation of non-viral lipidbased gene delivery is the toxicity of the cationic lipids that comprisethe non-viral delivery vehicle. The in vivo toxicity of liposomespartially explains the .discrepancy between in vitro and in vivo genetransfer results. Another factor contributing to this contradictory datais the difference in lipid vehicle stability in the presence and absenceof serum proteins. The interaction between lipid vehicles and serumproteins has a dramatic impact on the stability characteristics of lipidvehicles (Yang and Huang, 1997). Cationic lipids attract and bindnegatively charged serum proteins. Lipid vehicles associated with serumproteins are either dissolved or taken up by macrophages leading totheir removal from circulation. Current in vivo lipid delivery methodsuse subcutaneous, intradermal, intratumoral, or intracranial injectionto avoid the toxicity and stability problems associated with cationiclipids in the circulation. The interaction of lipid vehicles and plasmaproteins is responsible for the disparity between the efficiency of invitro (Felgner et al., 1987) and in vivo gene transfer (Zhu el al.,1993; Philip et al., 1993; Solodin et al., 1995; Liu et al., 1995;Thierry et al., 1995; Tsukamoto et al., 1995; Aksentijevich et al.,1996).

Advances in lipid formulations have improved the efficiency of genetransfer in vivo (Templeton et al. 1997; WO 98/07408). A novel lipidformulation composed of an equimolar ratio of1,2-bis(oleoyloxy)-3-(trimethyl ammonio)propane (DOTAP) and cholesterolsignificantly enhances systemic in vivo gene transfer, approximately 150fold. The DOTAP:cholesterol lipid formulation forms unique structuretermed a “sandwich liposome”. This formulation is reported to “sandwich”DNA between an invaginated bi-layer or ‘vase’ structure. Beneficialcharacteristics of these lipid structures include a positive p,colloidal stabilization by cholesterol, two dimensional DNA packing andincreased serum stability. Patent Application Nos. 60/135,818 and60/133,116 discuss formulations that may be used with the presentdisclosure.

The production of lipid formulations often is accomplished by sonicationor serial extrusion of liposomal mixtures after (I) reverse phaseevaporation (II) dehydration-rehydration (III) detergent dialysis and(IV) thin film hydration. Once manufactured, lipid structures can beused to encapsulate compounds that are toxic (chemotherapeutics) orlabile (nucleic acids) when in circulation. Lipid encapsulation hasresulted in a lower toxicity and a longer serum half-life for suchcompounds (Gabizon et al., 1990). Numerous disease treatments are usinglipid based gene transfer strategies to enhance conventional orestablish novel therapies, in particular therapies for treatinghyperproliferative diseases.

iii. Cell-Permeating Peptides

The present disclosure contemplates fusing or conjugating acell-penetrating peptide (also called a cell delivery domain, or celltransduction domain) to the polynucleotide comprising a cellcycle-dependent promoter operatively linked to a suicide gene. Suchdomains are well known in the art and are generally characterized asshort amphipathic or cationic peptides and peptide derivatives, oftencontaining multiple lysine and arginine resides (Fischer, 2007). Ofparticular interest are the TAT sequence from HIV1, and poly-D-Arg andpoly-D-Lys sequences (e.g., dextrorotary residues, eight residues inlength).

B. Regulatory Elements

Expression cassettes included in vectors useful in the presentdisclosure in particular contain (in a 5′-to-3′ direction) a eukaryotictranscriptional promoter operably linked to a protein-coding sequence,splice signals including intervening sequences, and a transcriptionaltermination/polyadenylation sequence. The promoters and enhancers thatcontrol the transcription of protein encoding genes in eukaryotic cellsare composed of multiple genetic elements. The cellular machinery isable to gather and integrate the regulatory information conveyed by eachelement, allowing different genes to evolve distinct, often complexpatterns of transcriptional regulation. A promoter used in the contextof the present disclosure includes a cell-cycle dependent promoter.

1. Promoter/Enhancers

In certain embodiments, the expression constructs provided hereincomprise a cell-cycle dependent promoter to drive expression of thesuicide gene. A promoter generally comprises a sequence that functionsto position the start site for RNA synthesis. The best known example ofthis is the TATA box, but in some promoters lacking a TATA box, such as,for example, the promoter for the mammalian terminal deoxynucleotidyltransferase gene and the promoter for the SV40 late genes, a discreteelement overlying the start site itself helps to fix the place ofinitiation. Additional promoter elements regulate the frequency oftranscriptional initiation. Typically, these are located in the region30-110 bp upstream of the start site, although a number of promotershave been shown to contain functional elements downstream of the startsite as well. To bring a coding sequence “under the control of” apromoter, one positions the 5′ end of the transcription initiation siteof the transcriptional reading frame “downstream” of (i.e., 3′ of) thechosen promoter. The “upstream” promoter stimulates transcription of theDNA and promotes expression of the encoded RNA.

The spacing between promoter elements frequently is flexible, so thatpromoter function is preserved when elements are inverted or movedrelative to one another. In the tk promoter, the spacing betweenpromoter elements can be increased to 50 bp apart before activity beginsto decline. Depending on the promoter, it appears that individualelements can function either cooperatively or independently to activatetranscription. A promoter may or may not be used in conjunction with an“enhancer,” which refers to a cis-acting regulatory sequence involved inthe transcriptional activation of a nucleic acid sequence.

A promoter may be one naturally associated with a nucleic acid sequence,as may be obtained by isolating the 5′ non-coding sequences locatedupstream of the coding segment and/or exon. Such a promoter can bereferred to as “endogenous.” Similarly, an enhancer may be one naturallyassociated with a nucleic acid sequence, located either downstream orupstream of that sequence. Alternatively, certain advantages will begained by positioning the coding nucleic acid segment under the controlof a recombinant or heterologous promoter, which refers to a promoterthat is not normally associated with a nucleic acid sequence in itsnatural environment. A recombinant or heterologous enhancer refers alsoto an enhancer not normally associated with a nucleic acid sequence inits natural environment. Such promoters or enhancers may includepromoters or enhancers of other genes, and promoters or enhancersisolated from any other virus, or prokaryotic or eukaryotic cell, andpromoters or enhancers not “naturally occurring,” i.e., containingdifferent elements of different transcriptional regulatory regions,and/or mutations that alter expression. For example, promoters that aremost commonly used in recombinant DNA construction include theβ-lactamase (penicillinase), lactose and tryptophan (trp) promotersystems. In addition to producing nucleic acid sequences of promotersand enhancers synthetically, sequences may be produced using recombinantcloning and/or nucleic acid amplification technology, including PCR™, inconnection with the compositions disclosed herein (see U.S. Pat. Nos.4,683,202 and 5,928,906, each incorporated herein by reference).Furthermore, it is contemplated that the control sequences that directtranscription and/or expression of sequences within non-nuclearorganelles such as mitochondria, chloroplasts, and the like, can beemployed as well.

Naturally, it will be important to employ a promoter and/or enhancerthat effectively directs the expression of the DNA segment in theorganelle, cell type, tissue, organ, or organism chosen for expression.Those of skill in the art of molecular biology generally know the use ofpromoters, enhancers, and cell type combinations for protein expression,(see, for example Sambrook et al. 1989, incorporated herein byreference). The promoters employed may be constitutive, tissue-specific,inducible, and/or useful under the appropriate conditions to direct highlevel expression of the introduced DNA segment, such as is advantageousin the large-scale production of recombinant proteins and/or peptides.The promoter may be heterologous or endogenous.

Additionally any promoter/enhancer combination (as per, for example, theEukaryotic Promoter Data Base EPDB, through world wide web atepd.isb-sib.ch/) could also be used to drive expression. Use of a T3, T7or SP6 cytoplasmic expression system is another possible embodiment.Eukaryotic cells can support cytoplasmic transcription from certainbacterial promoters if the appropriate bacterial polymerase is provided,either as part of the delivery complex or as an additional geneticexpression construct.

For cell replacement therapy using precursor cells, it is preferablethat the gene is expressed under the control of a cell cycle-dependentpromoter. Examples of cell cycle-dependent promoters include, but arenot limited to those listed in Table 1. Cell cycle-dependent promotersmay be derived from genes involved in the cell cycle, such as genes withexpression in the G(2) phase and mitosis. Cell cycle-dependent promotersmay comprise synthetic promoters. For cell replacement therapy,progenitors should generally maintain migratory potential and integrateappropriately into degenerating regions.

TABLE 1 Cell cycle-dependent promoters. Promoter References Ki-67 ZambonA.C. Cytometry A. 77(6):564-570, 2010. PCNA Whitfield et al., NatureReviews Cancer 6:99- CKS2 106, 2006. TOP2A BUB1, BUB1B CHEK1 AURKA,AURKB TRIP, TRIP13 CDC7 ORC1L PRIM1 RFC1 RRM1, RRM2 FEN1 CCNA2, CCNB1,CCNE1, CCNF CDC20 DDX11 E2F3 PKMYT1 PLK1 TIMP1 CDC25C CENPF, CENPN MCM2,MCM3, MCM4, MCM5, MCM6, MCM7, MCM10 E2F Dimova et al., Oncogene24:2810-2826, 2005. SKP2 Imaki et al., Cancer Res. 63(15):4607-13, 2003.CHR Muller et al., Nucleic Acids Res. 40(4):1561- 1578, 2012. SMC4 Renet al., Genes and Dev. 16:245-256, 2002. MELK Fischer et al., NucleicAcids Research 2015. CKAP2 Kang et al., Biochem Biophys Res Commun.420(4):822-7, 2012. DBF4 Wu X. and Lee H. Oncogene 21(51): 7786-96,2002. CDK4 Pawar et al. Oncogene 23(36):6125-35, 2004. ZWILCH Salvatoreet al., Cancer Res. 67(21):10148- POLE2 10158, 2007. ZWINT GINS2 SMC4HMMR NCAPH TTK PBK CEP55 ECT2 Seguin et al., Molecular and CellularBiology 29(2), 2009. STIL FBXO5 Balciunaite et al., Molecular andCellular Biology 25(18), 2005. SHCBP1 Grant et al., Molecular Biology ofthe Cell KIF23, KIF11, KIF4A 24(23):3634-3650, 2013. DLGAP5 RRM2 Zhanget al., Molecular Cancer 8(11), 2009. CDCA7 Gill et al., Molecular andCellular Biology 33(3):498-513, 2013. HELLS Mjelle et al., DNA Repair30:53-67, 2015. SGOL2 Llano et al., Genes Dev. 22(17):2400-13, 2008.KIAA0101/p15^(PAF) Chang et al., PLOS One 8(4):e61196, 2013. NUF2 Suzukiet al., Nature Cell Biology 18:382-392, NDC80 2016. NUSAP1 Yamamoto etal., Nucleic Acid Res. 2016. DTL Westendorp et al., Nucleic AcidsResearch 1-13, MLF1IP 2011. ASPM Wu et al., J Biol Chem.3(43):29396-29404, 2008.

In certain aspects, methods of the disclosure also concern enhancersequences, i.e., nucleic acid sequences that increase a promoter'sactivity and that have the potential to act in cis, and regardless oftheir orientation, even over relatively long distances (up to severalkilobases away from the target promoter). However, enhancer function isnot necessarily restricted to such long distances as they may alsofunction in close proximity to a given promoter.

2. Initiation Signals and Linked Expression

A specific initiation signal also may be used in the expressionconstructs provided in the present disclosure for efficient translationof coding sequences. These signals include the ATG initiation codon oradjacent sequences. Exogenous translational control signals, includingthe ATG initiation codon, may need to be provided. One of ordinary skillin the art would readily be capable of determining this and providingthe necessary signals. It is well known that the initiation codon mustbe “in-frame” with the reading frame of the desired coding sequence toensure translation of the entire insert. The exogenous translationalcontrol signals and initiation codons can be either natural orsynthetic. The efficiency of expression may be enhanced by the inclusionof appropriate transcription enhancer elements.

In certain embodiments, the use of internal ribosome entry sites (IRES)elements are used to create multigene, or polycistronic, messages. IRESelements are able to bypass the ribosome scanning model of 5′ methylatedCap dependent translation and begin translation at internal sites(Pelletier and Sonenberg, 1988). IRES elements from two members of thepicornavirus family (polio and encephalomyocarditis) have been described(Pelletier and Sonenberg, 1988), as well an IRES from a mammalianmessage (Macejak and Sarnow, 1991). IRES elements can be linked toheterologous open reading frames. Multiple open reading frames can betranscribed together, each separated by an IRES, creating polycistronicmessages. By virtue of the IRES element, each open reading frame isaccessible to ribosomes for efficient translation. Multiple genes can beefficiently expressed using a single promoter/enhancer to transcribe asingle message (see U.S. Pat. Nos. 5,925,565 and 5,935,819, each hereinincorporated by reference).

Additionally, certain 2A sequence elements could be used to createlinked- or co-expression of genes in the constructs provided in thepresent disclosure. For example, cleavage sequences could be used toco-express genes by linking open reading frames to form a singlecistron. An exemplary cleavage sequence is the F2A (Foot-and-mouthdisease virus 2A) or a “2A-like” sequence (e.g., Thosea asigna virus 2A;T2A) (Minskaia and Ryan, 2013).

3. Origins of Replication

In order to propagate a vector in a host cell, it may contain one ormore origins of replication sites (often termed “on”), for example, anucleic acid sequence corresponding to oriP of EBV as described above ora genetically engineered oriP with a similar or elevated function inprogramming, which is a specific nucleic acid sequence at whichreplication is initiated. Alternatively a replication origin of otherextra-chromosomally replicating virus as described above or anautonomously replicating sequence (ARS) can be employed.

4. Selection and Screenable Markers

In some embodiments, cells containing a construct of the presentdisclosure may be identified in vitro or in vivo by including a markerin the expression vector. Such markers would confer an identifiablechange to the cell permitting easy identification of cells containingthe expression vector. Generally, a selection marker is one that confersa property that allows for selection. A positive selection marker is onein which the presence of the marker allows for its selection, while anegative selection marker is one in which its presence prevents itsselection. An example of a positive selection marker is a drugresistance marker.

Usually the inclusion of a drug selection marker aids in the cloning andidentification of transformants, for example, genes that conferresistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin andhistidinol are useful selection markers. In addition to markersconferring a phenotype that allows for the discrimination oftransformants based on the implementation of conditions, other types ofmarkers including screenable markers such as GFP, whose basis iscolorimetric analysis, are also contemplated. Alternatively, screenableenzymes as negative selection markers such as herpes simplex virusthymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may beutilized. One of skill in the art would also know how to employimmunologic markers, possibly in conjunction with FACS analysis. Themarker used is not believed to be important, so long as it is capable ofbeing expressed simultaneously with the nucleic acid encoding a geneproduct. Further examples of selection and screenable markers are wellknown to one of skill in the art.

C. Suicide Genes and Prodrugs

In some embodiments, a suicide gene is a nucleic acid which, uponadministration of a prodrug, effects transition of a gene product to acompound which kills its host cell. Examples of suicide gene/prodrugcombinations which may be used are cytomegalovirus (CMV)-UL97, HerpesSimplex Virus-thymidine kinase (HSV-tk) and ganciclovir, acyclovir, orFIAU; oxidoreductase and cycloheximide; cytosine deaminase and5-fluorocytosine; thymidine kinase thymidilate kinase (Tdk::Tmk) andAZT; and deoxycytidine kinase and cytosine arabinoside. The E. colipurine nucleoside phosphorylase which converts the prodrug6-methylpurine deoxyriboside to toxic purine 6-methylpurine may also beused.

In some embodiments, the HSV thymidine kinase (TK) or CMV-UL97 suicidegene may have one or more mutations from the wild-type or unmutated HSVTK or CMV-UL97. As utilized herein, it should be understood that“unmutated thymidine kinase” refers to native or wild-type thymidinekinase such as that described by McKnight et al. (Nucl. Acids Res.8:5949-5964, 1980). The TK mutants can have one or more amino acidsubstitutions at residues 159-161 and 168-169 of HSV TK (Black et al.,Proc. Nat'l Acad. USA 93:3525-3529, 1996). The TK mutant used in thepresent methods may be SR11, SR26, SR39, SR4, SR15, SR32, or SR53 TKmutants. For example, the HSV TK can be HSV1-SR39TK (i.e, ₁₅₉₁FL₁₆₁ and₁₆₈FM₁₆₉, amino acid substitution at position 159 is leucine toisoleucine, at position 160 is isoleucine to phenylalanine, at position161 is phenylalanine to leucine, at position 168 is alanine tophenylalanine, and at position 169 is leucine to methionine) which hasan enhanced ability to convert the prodrug acyclovir and/or ganciclovirinto cytotoxic agents (U.S. Patent Publication Nos. 20130011903 and20120142071; incorporated herein by reference in their entirety).

The mutations may be upstream or downstream of the DRH nucleosidebinding site. For example, mutations which encode one or more amino acidsubstitutions from 1 to 7 amino acids upstream from the DRH nucleosidebinding site are contemplated. The biological activity of such kinasesmay be readily determined utilizing any of the assays which aredescribed herein, including for example, determination of the rate ofnucleoside analogue uptake, determination of the rate of nucleoside ornucleoside analogue phosphorylation. In addition, thymidine kinasemutants may be readily selected which are characterized by otherbiological properties, such as thermostability, and protein stability(e.g., described in U.S. Pat. No. 6,451,571).

Briefly, thymidine kinase mutants of the present disclosure may beprepared from a wide variety of Herpesviridae thymidine kinases,including for example both primate herpesviruses, and nonprimateherpesviruses such as avian herpesviruses. Representative examples ofsuitable herpesviruses include Herpes Simplex Virus Type 1 (McKnight etal., Nuc. Acids Res 8:5949-5964, 1980), Herpes Simplex Virus Type 2(Swain and Galloway, J. Virol. 46:1045-1050, 1983), Varicella ZosterVirus (Davison and Scott, J. Gen. Virol. 67:1759-1816, 1986), marmosetherpesvirus (Otsuka and Kit, Virology 135:316-330, 1984), felineherpesvirus type 1 (Nunberg et al., J. Virol. 63:3240-3249, 1989),pseudorabies virus (Kit and Kit, U.S. Pat. No. 4,514,497, 1985), equineherpesvirus type 1 (Robertson and Whalley, Nuc. Acids Res.16:11303-11317, 1988), bovine herpesvirus type 1 (Mittal and Field, J.Virol 70:2901-2918, 1989), turkey herpesvirus (Martin et al., J. Virol.63:2847-2852, 1989), Marek's disease virus (Scott et al., J. Gen. Virol.70:3055-3065, 1989), herpesvirus saimiri (Honess et al., J. Gen. Virol.70:3003-3013, 1989) and Epstein-Barr virus (Baer et al., Nature (London)310:207-311, 1984).

Such herpesviruses may be readily obtained from commercial sources suchas the American Type Culture Collection (“ATCC”, Rockville, Md.).Deposits of certain of the above-identified herpesviruses may be readilyobtained from the ATCC, for example: ATCC No. VR-539 (Herpes simplextype 1); ATCC Nos. VR-734 and VR-540 (Herpes Simplex type 2); ATCC No.VR-586 (Varicella Zoster Virus); ATCC No. VR-783 (Infectiouslaryngothracheitis); ATCC Nos. VR-624 VR-987, VR-2103, VR-2001, VR-2002,VR-2175, VR-585 (Marek's disease virus); ATCC Nos. VR-584B and VR-584B(turkey herpesvirus); ATCC Nos. VR-631 and VR-842 (bovine herpesvirustype 1); and ATCC Nos. VR-2003, VR-2229 and VR-700 (equine herpesvirustype 1). Herpesviruses may also be readily isolated and identified fromnaturally occurring sources (e.g., from an infected animal).

Thymidine kinase mutants used in the present disclosure may beconstructed using a wide variety of techniques. For example, mutationsmay be introduced at particular loci by synthesizing oligonucleotidescontaining a mutant sequence, flanked by restriction sites enablingligation to fragments of the native sequence. Following ligation, theresulting reconstructed sequence encodes a derivative having the desiredamino acid insertion, substitution, or deletion.

Alternatively, oligonucleotide-directed site-specific (or segmentspecific) mutagenesis procedures may be employed to provide an alteredgene having particular codons altered according to the substitution,deletion, or insertion required. Deletion or truncation derivatives ofthymidine kinase mutants may also be constructed by utilizing convenientrestriction endonuclease sites adjacent to the desired deletion.Subsequent to restriction, overhangs may be filled in, and the DNAreligated. Exemplary methods of making the alterations set forth aboveare disclosed by Sambrook et al. (Molecular cloning: A LaboratoryManual, 2d Ed., Cold Spring Harbor Laboratory Press, 1989).

Thymidine kinase mutants may also be constructed utilizing techniques ofPCR mutagenesis, chemical mutagenesis (Drinkwater and Klinedinst, PNAS83:3402-3406, 1986), by forced nucleotide misincorporation (e.g., Liaoand Wise Gene 88:107-111, 1990), or by use of randomly mutagenizedoligonucleotides (Horwitz et al., Genome 3:112-117, 1989).

In some embodiments, the suicide gene is the CMV-UL97 gene or a variantthereof. The prodrug may be ganciclovir, acyclovir, or penciclovir.

TABLE 2 Suicide Genes and Prodrugs (Denny et al., BiomedicineBiotechnol. 1:48-70, 2003). Suicide Gene Prodrug HSV thymidine kinase(TK) Ganciclovir (GCV) Ganciclovir elaidic acid ester Penciclovir (PCV)Acyclovir (ACV) Valacyclovir (VCV) (E)-5-(2-bromoviny])-2′- deoxyuridine(BVDU) Zidovuline (AZT) 2′-exo-methanocarbathymidine (MCT) CytosineDeaminase (CD) 5-fluorocytosine (5-FC) Purine nucleoside phosphorylase(PNP) 6-methylpurine deoxyriboside (MEP) fludarabine (FAMP) Cytochromep450 enzymes (CYP) Cyclophosphamide (CPA) Ifosfamide (IFO) 4-ipomeanol(4-IM) Carboxypeptidases (CP) 4-[(2-chloroethyl)(2- mesyloxyethyl)amino]benzoyl-L-glutamic acid (CMDA) Hydroxy- and amino-aniline mustardsAnthracycline glutamates Methotrexate α-peptides (MTX-Phe) Caspase-9AP1903 (Di Stasi et al., 2011) Carboxylesterase (CE) Irinotecan (IRT)Anthracycline acetals Nitroreductase (NTR) dinitroaziridinylbenzamideCB1954 dinitrobenzamide mustard SN23862 4-Nitrobenzyl carbamatesQuinones Horse radish peroxidase (HRP) Indole-3-acetic acid (IAA)5-Fluoroindole-3-acetic acid (FIAA) Guanine Ribosyltransferase (XGRTP)6-Thioxanthine (6-TX) Glycosidase enzymes HM1826 Anthracycline acetalsMethionine-α,γ-lyase (MET) Selenomethionine (SeMET) Thymidinephosphorylase (TP) 5′-Deoxy-5-fluorouridine (5′-DFU)

IV. METHODS OF USE

PSCs to which genes have been introduced by vectors of the presentdisclosure (e.g., pseudotyped lentiviral vectors), and cells, tissues,organs and such differentiated from these PSCs are useful for assayingand screening for various types of pharmaceutical agents. Through genetransfer into PSCs, for example, pharmaceutical agents or genes forcarrying out specific differentiation of tissues or cells, andparticularly preferably tissues or cells derived from primates, can beevaluated for their effects or screened for.

The present disclosure also encompasses PSCs into which vectors of thepresent disclosure (e.g., pseudotyped lentiviral vectors) have beenintroduced, and differentiated cells and tissues that havedifferentiated from the PSCs. The differentiated cells and tissues canbe identified based on marker expression and morphologicalcharacteristics specific to the tissues or cells.

A. Methods of Treatment

In some embodiments, the present disclosure provides methods of treatinga disease or disorder in a subject comprising administering a populationof precursor cells which comprise a suicide gene under the control of acell cycle-dependent promoter. The precursor cells may be used toreplace cells which are essentially non-dividing cells and any remainingcycling or proliferating cells may be eliminated by the administrationof a pro-drug which is selectively kills these cycling cells. Thus, themethod can prevent the formation of teratomas or tumors from theremaining cycling cells.

The treatment methods may be applied to any disease or disorder whichmay benefit from the replacement of a certain cell population. Forexample, neuronal diseases may be treated by the administration ofprecursor cells. Disease which affect the vasculature, such as tumorangiogenesis, may be treated by the administration of endothelialprecursor cells. Cardiomyocyte precursor cells may be used in thetreatment of heart diseases and pancreatic precursor cells may be usedfor the treatment of pancreatic diseases. Other precursor cell therapiesof the present disclosure may comprise, but are not limited to, kidneyprecursor cells, oligodendrocyte precursor cells, hematopoieticprecursor cells, myeloid precursor cells, mesenchymal precursor cells,retinal precursor cells, and osteoclast precursor cells.

In humans, there are numerous diseases affecting the CNS, many of whichresult in cerebellar degeneration with concomitant symptoms, such asdysmetria, ataxia, past pointing, dysdiadochokinesia, dysarthria,intention and action tremor, cerebellar nystagmus, rebound, hypotonia,and loss of equilibrium. These diseases may be alleviated using cellreplacement therapy according to the specific embodiment disclosedherein.

Diseases of the CNS and brain in humans that are amenable to treatmentusing the methods of the present disclosure include a wide variety ofdiseases and disorders, including for example, Huntington's disease;Alzheimer's disease (both sporadic and familial); Parkinson's diseaseand Parkinson's disease-like symptoms, such as muscle tremors, muscleweakness, rigidity, bradykinesia, alterations in posture andequilibrium, and dementia; amyotrophic lateral sclerosis (ALS); spinalcord injury; severe epilepsy; traumatic brain injury; and the like.

Accordingly, the methods of the present disclosure may be used toalleviate abnormalities of the CNS and cerebellum that result indemyelination, dysmyelination, dementia, dysmetria, ataxia, pastpointing, dysdiadochokinesia, dysarthria, intention and action tremor,cerebellar nystagmus, rebound, hypotonia, and loss of equilibrium. It iswell established that patients with Parkinson's suffer fromprogressively disabled motor control due to loss of dopaminergic neuronswithin the basal ganglia, which innervate the striatum. Neural precursorcells can be directed toward a dopaminergic fate and delivered into thestriatum using the systems described herein to prevent overgrowth andtumor formation. Likewise, neural precursors of the present disclosurecan be directed toward a cholinergic fate for use in the treatment ofAlzheimer's disease.

The methods of the present disclosure also have use in the veterinaryfield including treatment of domestic pets and farm animals. As utilizedherein, the terms “treated, prevented, or, inhibited” refer to thealteration of a disease course or progress in a statisticallysignificant manner. Determination of whether a disease course has beenaltered may be readily assessed in a variety of model systems and byusing standard assays, known in the art, which analyze the ability of agene delivery vector to delay or prevent CNS or cerebellar degeneration.

Gene delivery vectors may be delivered directly to the CNS or brain byinjection into, e.g., a ventricle, a cerebellar lobule and/or thestriatum, using a needle, catheter or related device. In particular,within certain embodiments of the present disclosure, one or moredosages may be administered directly in the indicated manner at dosagesgreater than or equal to 103, 104, 105, 106, 107, 108, 109, 1010 or 1011cfu. Cerebellar injections are complicated by the fact that stereotaxiccoordinates cannot be used to precisely target the site of an injection;there is animal to animal variation in the size of cerebellar lobules,as well as their absolute three-dimensional orientation. Thus, choleratoxin subunit b (CTb) may be used to determine the exact location of theinjection and reveal the pool of transducable neurons at an injectionsite. Injections may fill the molecular layer, Purkinje cell layer,granule cell layer and white matter of the arbor vitae but do not extendto the deep cerebellar nuclei.

Alternatively, and preferably for treating diseases using transducedneural progenitor cells, neural progenitor cells are first transduced exvivo and then delivered to the CNS. Generally, if transduced ex vivo,cells will be infected with the viral vectors described herein at an MOIof about 0.01 to about 50, preferably about 0.05 to about 30, and mostpreferably about 0.1 to about 20 MOI. For FIV vectors, an MOI of about0.05 to about 10, preferably about 0.1 to about 5, or even 0.1 to about1, should be sufficient. Once transfected ex vivo, cells can bedelivered, for example, to the ventricular region, as well as to thestriatum, spinal cord and neuromuscular junction, using neurosurgicaltechniques known in the art, and as described in the examples below,such as by stereotactic injection and injections into the eyes and ears(see, e.g., Stein et al., J Virol 73:3424-3429, 1999; Davidson et al.,PNAS 97:3428-3432, 2000; Davidson et al., Nat. Genet. 3:219-223, 1993;and Alisky and Davidson, Hum. Gene Ther. 77:2315-2329, 2000). Ingeneral, the amount of transduced cells in the compositions to bedelivered to the subject will be from about 101 to about 1010 cells ormore, more preferably about 101 to 108 cells or more, and even morepreferably about 102 to about 104 cells, or more. Other effectivedosages can be readily established by one of ordinary skill in the artthrough routine trials establishing dose response curves.

A wide variety of assays may be utilized in order to determineappropriate dosages for administration, or to assess the ability of agene delivery vector to treat or prevent a particular disease. Certainof these assays are discussed in more detail below. For example, theability of particular vectors to transduce cerebellar neurons and neuralprogenitor cells can be assessed using reporter genes, as discussedbelow. The ability of the transduced progenitor cells to differentiatemay be tested, for example, using immunocytochemistry, as discussedbelow in the examples.

For systemic administration, a therapeutically effective dose can beestimated initially from in vitro assays. For example, a dose can beformulated in animal models to achieve a circulating concentration rangethat includes the IC₅₀ as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Thetherapy may be repeated intermittently while symptoms detectable or evenwhen they are not detectable. The therapy may be provided alone or incombination with other drugs.

Initial dosages can also be estimated from in vivo data, e.g., animalmodels, using techniques that are well known in the art. One havingordinary skill in the art could readily optimize administration tohumans based on animal data.

B. Pharmaceutical Preparations

Where clinical application of a composition containing a therapeuticcell population of the present disclosure is undertaken, it willgenerally be beneficial to prepare a pharmaceutical compositionappropriate for the intended application. This will typically entailpreparing a pharmaceutical composition that is essentially free ofpyrogens, as well as any other impurities that could be harmful tohumans or animals.

The phrases “pharmaceutical or pharmacologically acceptable” refers tomolecular entities and compositions that do not produce an adverse,allergic or other untoward reaction when administered to an animal, suchas a human, as appropriate. The preparation of a pharmaceuticalcomposition comprising a inhibitory nucleic acid or additional activeingredient will be known to those of skill in the art in light of thepresent disclosure, as exemplified by Remington (2005), incorporatedherein by reference. Moreover, for animal (e.g., human) administration,it will be understood that preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biological Standards.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, gels, binders, excipients, disintegration agents,lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art. A pharmaceutically acceptable carrier is particularlyformulated for administration to a human, although in certainembodiments it may be desirable to use a pharmaceutically acceptablecarrier that is formulated for administration to a non-human animal butwhich would not be acceptable (e.g., due to governmental regulations)for administration to a human. Except insofar as any conventionalcarrier is incompatible with the active ingredient, its use in thetherapeutic or pharmaceutical compositions is contemplated.

The present therapies of the embodiments can be administeredintravenously, intradermally, intraarterially, intraperitoneally,intralesionally, intracranially, intraarticularly, intraprostaticaly,intrapleurally, intratracheally, intranasally, intravitreally,intravaginally, intrarectally, topically, intratumorally,intramuscularly, intraperitoneally, subcutaneously, subconjunctival,intravesicularlly, mucosally, intrapericardially, intraumbilically,intraocularally, orally, topically, locally, inhalation (e.g., aerosolinhalation), injection, infusion, continuous infusion, localizedperfusion bathing target cells directly, via a catheter, via a lavage,in cremes, in lipid compositions (e.g., liposomes), or by other methodor any combination of the forgoing as would be known to one of ordinaryskill in the art

The actual dosage amount of a composition of the present disclosureadministered to a patient or subject can be determined by physical andphysiological factors such as body weight, severity of condition, thetype of disease being treated, previous or concurrent therapeuticinterventions, idiopathy of the patient and on the route ofadministration. The practitioner responsible for administration will, inany event, determine the concentration of active ingredient(s) in acomposition and appropriate dose(s) for the individual subject. Innon-limiting examples, a dose may comprise from about 5microgram/kg/body weight to about 500 milligram/kg/body weight, about 5mg/kg/body weight to about 100 mg/kg/body weight, about 10 mg/kg/bodyweight to about 50 mg/kg/body weight, can be administered.

V. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples that follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments that are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1— Targeting of Proliferative Cells

Generation of TK-PSC line: The aim of this study was to develop asuicide gene approach towards the prevention of uncontrolled overgrowthof PSC-derived transplanted neural cells. For many reasons, ganciclovirand herpes simplex thymidine kinase is an attractive system for such asuicide gene technology. Indeed, ganciclovir is widely used in patientsand penetrates into the central nervous system. However, constitutiveexpression of HSV-TK would not be desirable, as this could lead toimmune rejection of transplanted cells, and the use of ganciclovir couldpotentially lead to killing of functional transplanted neurons.

Accordingly, HSV-TK was expressed under the control of a cellcycle-dependent Ki67 promoter fragment. For this purpose the humanpluripotent ESC (hPSC) line HS415 was transduced with a lentivectorcoding for the expression of HSV-TK under the control of the Ki67promoter fragment (Zambon, 2010). As the lentivector transductionefficiency of hPSC is in the range of 10-20%, a tool for selection oftransduced cells was required. Thus, the construct that was used had theHSV-TK sequence fused to a zeocin resistance sequence. HSV-TK-positivecells were selected by culturing transduced hESC in the presence ofzeocin (FIG. 1A). A polyclonal HSV-TK-expressing PSC line was obtained,referred to herein as TK-PSC. Neural precursor cells and mature neuronswere obtained from TK-PSC and are to as TK-NPC and TK-neurons,respectively (FIG. 1B, 1C).

Highly proliferative cells expressing Ki67/TK are sensitive toganciclovir in vitro: Next, TK expression was investigated in TK-PSC andin TK neurons. In TK-PSC, virtually 100% of cells expressed TK inconcordance with high expression of Ki67 in these cells (FIG. 1C, upperpanel). In contrast, TK neurons did not express any TK, as predictedfrom them being post-mitotic cells. However, there were stillKi67-positive cells in the neuronal preparation (FIG. 1C, lower panel).These Ki67-positive cells likely do not reflect the presence ofproliferating cells as many of the Ki67-positive cells in the neuronalpreparation clearly showed a morphology of mature neurons with longneurite extension. The expression of Ki67 was also investigated,together with expression of the pluripotency markers nanog and oct3/4 byflow cytometry. In PSCs, virtually all cells were Ki67-, nanog-, andoct3/4-positive. In contrast, NPCs rapidly lost pluripotency markersafter 1 week of neurosphere differentiation, while Ki67 expressiondecreased more slowly with levels slightly above background after 3weeks of differentiation (FIG. 2A).

The impact of ganciclovir on TK cells was determined in vitro (FIG. 2B,2C, 2D). TK PSCs were highly sensitive to ganciclovir exposure (96 h)and even at ganciclovir concentrations of 2.5 μM loss of cells wasobserved, which was almost complete at 10 μM (FIG. 2B, 2C). In contrast,regular PSCs (i.e., not transduced with the TK construct) were resistantto ganciclovir even at concentrations of 40 μM (FIG. 2B, 2F). Asexpected from the lack of TK expression in TK neurons, TK neurons werenot affected by ganciclovir concentrations up to 40 μM, similar to thecontrol neurons.

The time course of the ganciclovir effect was also investigated (FIG.2D). TK PSCs were killed by Ganciclovir (40 μM) within 4 days. TK-NPCsafter 1 week of neurosphere differentiation were still sensitive toGanciclovir (40 μM), however the time course of killing was markedlyslowed down and complete killing was observed only after 8 days. TK-NPCsafter 2 weeks of neurosphere differentiation showed only little cellgrowth and were not killed by ganciclovir.

Early, but not late ganciclovir treatment prevents tumor formation upontransplantation of highly proliferative pluripotent stem cells: Giventhe encouraging results in vitro, it was investigated whetherganciclovir could prevent tumor formation in vivo. For this purpose,TK-PSC were transplanted into the striatum of NOD/SCID mice. In theabsence of ganciclovir, mice, sacrificed 49 days post transplantation,consistently developed teratomas (FIG. 4A), which consisted of humancells (HCM-positive) and showed abundant expression of Ki67 and Ki67promoter-driven HSV-TK (FIG. 5Aa). There was a moderate amount of mousemicroglia invasion. In contrast, mice that were treated for 15 days withganciclovir (starting 4 days post-transplantation) had no observedteratomas (FIG. 4B). There were hardly any human cells left in theganciclovir-treated animals, and the few surviving human cells werenegative for HSV-TK and—to a large extent—also negative for Ki67,suggesting that the few surviving human cells had become post-mitoticand therefore lost sensitivity to ganciclovir (FIG. 5Ba). Thus,ganciclovir prevented teratoma formation by transplanted TK-PSC.

It was next investigated whether ganciclovir could also be used to treatalready established teratomas. Therefore, teratoma formation was allowedto progress for 30 days (preliminary results had shown that within thistime delay there was consistent teratoma formation upon transplantationof PSC) and treated with ganciclovir (or PBS) for days 30-45post-transplantation. Mice were sacrificed 30 days after the end of theganciclovir treatment. Under these conditions, there was teratomaformation in both, the PBS and the ganciclovir-treated animals (FIG. 6). CD31 staining showed that tumors were vascularized, suggesting thatlack of perfusion did not account for the absence of a ganciclovirtherapeutic effect in established tumors. Similarly, many teratoma cellsshowed high level expression of HSV-TK suggesting that down-regulationof the transgene was not the explanation for the lack of a therapeuticresponse.

A previous study (Chalmers et al., 2001) had suggested that alternativesplicing, due to cryptic splice donor and acceptor sites, can lead toformation of an inactive TK. Another study (Kotini et al., 2016) hadsuggested that various non-sense mutations may occur in the TK sequencein rapidly growing cells and might explain ganciclovir resistance.Therefore, mRNA was extracted from the tumors formed upon injection ofundifferentiated pluripotent cells. From these mRNA preparations, fiveregions of the TK protein were amplified covering a total of 1162nucleotides, corresponding to 85% of the total TK sequence (FIG. 7A).The sequenced regions also included the proposed cryptic splice sites(e.g., amplicon 3). The PCR products were directly sequenced (FIG. 7B).The results of this sequencing determined that the all over sequencefound in the tumors was identical to the plasmid HSV-TK sequence,suggesting that—if mutations had occurred—they were not representativeof the majority of HSV-TK sequences within the tumor. Inspection of thesequencing results did not show any evidence for ambivalent sequencingsignals. In addition, the cryptic splice sites described previously werenot present in this version of the sequence (neither in the plasmidsequence nor in the tumor-derived sequences); accordingly, no splicevariant was detected in the sequencing results.

To investigate whether minority sequences with mutations had occurredwithin the tumor, two of the PCR amplicons were subcloned and sequenced.No mutation was observed in subclones of amplicon 5; in contrast, in twoof the subclones of amplicon 3, there was a single point mutation (FIG.7C) not located at critical sites of the TK enzyme (ATP andnucleotide-binding sites) (Andrei et al., 2005).

Transplantation of TK-NPCs produced mature neurons which were notsensitive to ganciclovir treatment: The in vivo results, shown so far,were obtained with transplantation of undifferentiated pluripotent stemcells. However, the final goal of neuronal cell therapy is thetransplantation of neural precursor cells, which should differentiateinto mature neurons and be resistant to ganciclovir treatment.Therefore, 2-3 weeks-NPC containing neurospheres were transplanted(FIGS. 3C, 8 ). Under these conditions, no tumor formation was observed,even in the absence of ganciclovir treatment (FIG. 8A). In line withthese observations, the transplanted cells (assessed byimmunofluorescence 7 weeks after transplantation) were abundantlypositive for beta 3 tubulin (FIG. 8B, upper panel), while a smallerfraction of cells was TH-positive (FIG. 8B, lower panel). Occasionallynestin-positive cells were observed. None of these markers were affectedby ganciclovir treatment.

Markers of cellular proliferation were analyzed by staining of thetransplants with antibodies against PCNA and Ki67 (FIG. 8C).Surprisingly, many of the transplanted cells were PCNA positive, whileKi67 staining of the transplants was low to absent. Ganciclovirtreatment did not alter this pattern (FIG. 8C, left panels, control andright panels, ganciclovir treatment). To verify whether thePCNA-positive, Ki67-negative cells were proliferating cells, BrdUexperiments were performed. No significant BrdU staining was observed(FIG. 8C, insert), suggesting that the absence of Ki67 correctlyindicated post-mitotic cells, and that PCNA expression may persist evenafter proliferation arrest.

To compare tumor formation and response to ganciclovir under differentexperimental conditions, graft surfaces were quantified (FIG. 8D). WhenPSCs were transplanted, large tumors (graft surface ˜10-15 mm2)developed, which was completely prevented by early ganciclovirtreatment, while late ganciclovir treatment did not have any effect ontumor size. Finally, upon transplantation of NPC (derived from 2-3 weeksold neurospheres), small non-tumoral transplants (graft surface ˜2-3 mm)were observed. The size of these transplants was not affected byganciclovir.

Thus, the present methods provide a novel tool that allows in vivoremoval of proliferating, potentially tumorigenic cells from neuraltransplants. The system is based on the expression of HSV-TK under thecontrol of the cell cycle-dependent Ki67 promoter. It will beparticularly useful for the transplantation of pluripotent stemcell-derived neurons. Indeed, HSV-TK expressing cells can be eliminatedby treatment of patients with the clinically used, CNS-permeant drugganciclovir. As the Ki67 promoter will be inactive in mature neurons,treatment with ganciclovir will only eliminate proliferating precursors,but preserve the integrity of differentiated post-mitotic neurons.

Example 2—Materials and Methods

Culture of undifferentiated pluripotent stem cells: Human ES cell lineHS415 (used from passage 17 to 30, Outi Hovatta, Karolinska Institute,Stockholm, Sweden) was cultured onto human extracellular matrix (MAXGEL®ECM, dilution 1/50 from Sigma or on MATRIGEL™, dilution 1/100,Invitrogen) in a feeder-free culture medium (Nutristem from BiologicalIndustries). Medium was changed one another day to maintainpluripotency. Cells were passaged with enzymatic procedure (ACCUTASE®;Invitrogen) and replated with Rho-associated protein kinase (ROCK)inhibitor (10 μM Y-27632; Ascient Biosciences) during 24 h beforeremoval.

Lentiviral vector construction, cell transduction and selection:Construction of plasmids and lentiviral vectors: The final lentivectorplasmid was generated by an LR Clonase II (Invitrogen, Carlsbad,Calif.)-mediated recombination of a pENTR plasmid containing the HsKi67promoter (pENTR-L4-Ki67-L1R), a pENTR plasmid containing the fusion geneTK::Sh, corresponding to the thymidine kinase (TK) gene from Herpessimplex virus type 1 (HSV1) and the Shble gene conferring zeocinresistance, and a pCLX-R4-DEST-R2 lentivector destination cassette.

Lentiviral vector production and titration: Lentiviral vector stockswere generated using transient transfection of HEK 293T cells with thespecific lentivector transfer plasmid, the psPAX2 plasmid encodinggag/pol and the pCAG-VSVG envelope plasmid. Lentivector titer wasperformed using transduction of HT-1080 cells followed by flow cytometryquantification of GFP-positive cells 5 days after infection. Cellculture: HEK 293T and HT-1080 cells were cultured in high-glucoseDulbecco's modified eagle medium (Sigma) supplemented with 10% fetalcalf serum, 1% Penicillin, 1% Streptomycin, and 1% 1-glutamine.Transduction of HS415 cell line: 1 up to 5 copies of the lentiviralvector were introduced. HS415 cells were cultured for 5 days beforezeocin selection.

Generation of neurospheres containing DA neuropecursors: NPC weregenerated as neurospheres to contain DA progenitors as describedpreviously (Tieng et al., 2014). Briefly, neural midbrain orientation isperformed during the first week followed by 2 additional weeks ofmaturation to obtain 1 week or 2 or 3 week-old NPC (FIGS. 1B, 3A).

Generation of NPC in two dimensional culture (2D): For differentiationin 2D, 3-week old neurospheres were dissociated with ACCUMAX®(Millipore) and replated on polyornithine—(15 μg/mL; Sigma) and laminin-(2 m/cm²; R&D System) coated cover slips (diameter=0.8 cm) in 24-wellplates at 200,000 cells/cm² in maturation medium for 1 week beforeanalysis by immunofluorescence staining (FIG. 1B).

Flow cytometry: Undifferentiated ES cells were enzymatically detached assingle cells from human matrix (Accutase, Invitrogen), washed with PBSbefore a 10 min fixation step in 4% paraformaldehyde (PFA) at roomtemperature. One week- or 3 week-old neurospheres were dissociated(Accutase, Invitrogen). To detected intracellular antigens, cells werepermeabilized in 1× perm/wash buffer (BD bioscience) for 10 min, before30 min incubation in the dark at room temperature with differentantibodies (PE Nanog and PerCp-Cy 5.5 Oct 3/4 from Life Technology,FITC-ki67, rabbit, from Abcam) After washing, cells were immediately runor stored at 4° C. for 24 h maximum. For each sample run, 10,000 eventswere recorded and analyzed. Flow cytometry acquisition was performedusing FacsCanto I equipment with 488 and 633 lasers (BD bioscience) anddata analysis by Flowjo software.

Immunofluorescence staining of fixed cells: After 1 week of culture, 2Dcultures on cover slips were fixed in 4% PFA for 10 min at roomtemperature, washed and processed for conventional immunocytochemistry.Paraffin embedded brain was sectioned and processed with cresyl violetfor morphological assessment and immunohistochemistry staining. Primaryantibody was incubated at 4° C. overnight in agitation in PBS+0.1%triton for cells and 0.3% triton for tissue. Revelation is performedwith a secondary antibody at room temperature for 30 min.

Primary antibodies were against Nestin (rabbit polyclonal anti-human,from Chemicon; 1/400), β3-tubulin (mouse monoclonal from Sigma or rabbitpolyclonal from Covance; 1/2,000, neuronal nuclei-specific protein(NeuN, mouse monoclonal from Chemicon; 1/1,000), glial fibrillary acidicprotein (GFAP, rabbit polyclonal from Dako; 1/2,000), proliferating cellnuclear antigen (PCNA, mouse monoclonal from Dako; 1/100), tyrosinehydroxylase (TH; rabbit polyclonal from Millipore; 1/500). Detection ofprimary antibodies was performed using appropriate species-specificAlexa 488- or Alexa 555-labeled secondary antibodies. Controls includedexamination of the cell or tissue autofluorescence and omission of thefirst antibody. Cell nuclei were stained with DAPI. Sections and cellcultures were mounted in Fluorosave (Calbiochem) or Eukitt (KindlerGmbH) and observed with an Axioscop 2 plus microscope equipped withappropriate filters, Axiocam color camera, and Axiovision software(Leitz).

Cell proliferation measurement by calcein assay: Cells were plated ontoa 96 wells pre-coated with polyornothine+ECM (1/50). Respectively 1000cells for pluripotent stem cells, 5000 cells for early NPC and 30000cells for late NPC. Ganciclovir (Cemevene diluted in PBS, Roche)treatment was added the day after cell plating for PSC, 3 days later forearly NPC and one week later for late NPC. Calcein (2 μM) was added fordifferent time course of ganciclovir treatment (24H to 96H). Cellsurvival was measured by calcein integration after 24 h of incubation ina 37° C. incubator.

Stereotaxic engraftment and ganciclovir treatment: Undifferentiatedpluripotent stem cells (20000 cells/μL) and dissociated neurospheres(100.000 cells/μL) were injected in the striatum of anesthetized miceusing a 27-G Hamilton syringe. Injection coordinates for lateralstriatum were: bregma=0.74 mm, mediolateral=2.25 mm, dorsoventral=3.5mm; for medial striatum: bregma=0.74 mm, mediolateral=1.4 mm,dorsoventral=2.8 mm. Mice were euthanized one month later afterganciclovir termination.

Morphological and Phenotypic Analysis of Injected Surviving Cells:Anesthetized mice were fixed by intracardiac perfusion of 4%paraformaldehyde in phosphate-buffered saline. Immunohistochemicaldetection was performed on 10 μm thick free-floating cryostat sectionsusing Alexa 488-, or 555-labeled secondary antibodies or thebiotin-avidin-peroxidase complex method (Vector). The following primaryantibodies were used: Nestin (rabbit polyclonal anti-human, fromChemicon, 1/400), β3-tubulin (mouse monoclonal from Sigma or rabbitpolyclonal from Covance, 1/2000), Neuronal Nuclei-specific protein(NeuN, mouse monoclonal from Chemicon, 1/1000), Glial Fibrillary AcidicProtein (GFAP, rabbit polyclonal from Dako, 1/2000), Proliferating CellNuclear Antigen (PCNA, mouse monoclonal from Dako, 1/100), TyrosinHydroxylase (TH, rabbit polyclonal from millipore, 1/500), iba 1(polyclonal rabbit from Wako, 1/500), HSV-TK (mouse monoclonal, Gentaur,1/100), ki67 (monoclonal rabbit, Abcam, 1/100 or mouse, Chemicon,1/100). CD31 (Detection of primary antibodies was performed usingappropriate species specific Alexa 488- or Alexa 555-labeled secondaryantibodies. Controls included examination of the cell or tissueauto-fluorescence and omission of the first antibody. Cell nuclei werestained with DAPI. Sections and cells cultures were mounted inFluorosave (Calbiochem) or Eukitt (Kindler GmbH, Germany) and observedwith an Axioscop2 plus microscope equipped with appropriate filters,Axiocam color camera and Axiovision software (Leitz, Germany). Confocalimaging was achieved with an LSM 510 Meta confocal laser scanner andBitplane SS Imaris 5.7.2 software.

BrdU labelling: BrdU (100 mg/kg, Millipore) was injectedintraperitoneally twice daily for 3 consecutive days. Mice weresacrificed 5 days later and perfused, through the heart, with PBS and 4%paraformaldehyde in PBS. Brain and small intestine were embedded inparaffin and 10 mm thick frontal section were processed for BrdUimmunohistochemiscal detection using a BrdU Immunohistochemistry Kit(Chemicon, Cat No. 2760), small intestine section serving as control.

Thymidine kinase sequencing: Total RNA was extracted and purifiedaccording the manufacturers' instructions (high pure FFPE RNA micro kit,Roche) from PFA-fixed, paraffin-embedded tissue delimited to teratomaarea induced by PSC transplantation and late ganciclovir treatment (IG.7). Typically, RNA isolated from formalin-fixed tissue is fragmented andthe bulk of RNA obtained is around 200 bases in length. After DNasetreatment, CDNA synthesis is performed from RNA pool extracted (Takara).Thymidine kinase gene expressed inside the teratoma was amplified byusing a set of different overlapping primers along the thymidinesequence (1162 bp). PCR reaction is performed with a proofreading taqpolymerase (Q5 high fidelity, NEB) during 35 cycles with 30″ at 95° C.,30″ at 58° C. and 1 min at 72° C. Primers for fragment 1 are for forwardF 5′-GAG CGGTGGTTCGACAAGTGG-3′ (SEQ ID NO: 1) and reverse R5′-CCTCAGCAGGGTTGGCATC-3′ (SEQ ID NO: 2), fragment 2, F5′-GATGCCAACCCTGCTGAGG-3′ (SEQ ID NO: 3) and R 5′-GTCCAGCCTGTGCTGGGTG-3′(SEQ ID NO: 4), fragment 3, F 5′-CACCCAGCACAGGCTGGAC-3′ (SEQ ID NO: 5)and R 5′-CAGGGCCACAAAAGCCAGCAC-3′ (SEQ ID NO: 6), fragment 4, F5′-GTGCTGGCTTTTGTGGCCCTG-3′ (SEQ ID NO: 7) and R5′-CCAGAGAGCTGTCCCCAGTC-3′ (SEQ ID NO: 8), fragment 5, F5′-GTCCCCTGCTGGATGCAGAG-3′ (SEQ ID NO: 9) and R 5′-CTC TGCATCCAGCAGGGGAC-3′ (SEQ ID NO: 10). Fragment 1 to 5 were directlysequenced or subcloned into topo TA cloning kit (Invitrogen) beforeperforming sequencing of the different bacterial clones (Microsynth,AG).

Example 3—Suicide Gene Construct Characterization

In further experiments, cell proliferation assay was performed usingdifferent suicide gene constructs in combination with various drugs,such as acyclovir, ganciclovir, or pencilovir. In the first assay, akinase from human cytomegalovirus (CMV), UL97, was used as a novelsuicide gene. The HEK cells were transfected with a high copy number ofthe respective plasmids. It was observed that UL97 under the UBIpromoter plasmid when transfected in the HEK cells showed sensitivity toganciclovir (FIG. 9 ) similar to the HSV-TK and SR39 plasmids.

In another experiment, the cells were transduced with a low copy numberof the plasmids and, thus, differences between the different nucleosideanalogs could be observed. It was observed that ganciclovir killedHSV-TK and SR39-transduced cells at a relatively low concentration,while very high concentrations of acyclovir are needed (FIG. 10A).Ganciclovir at high concentrations is also toxic to non-transducedcells.

Finally, the cell proliferation assay was also performed with the SR39or HSV-TK-transduced cells treated with penciclovir. It was found thatpenciclovir kills the transduced cells at relatively low concentrationsand did not show any toxicity in untransduced cells (FIG. 10B). Thus,penciclovir may be used as the prodrug with the suicide gene construct,such as HSV-TK, SR39, or UL97.

All of the methods disclosed and claimed herein can be made and executedwithout undue experimentation in light of the present disclosure. Whilethe compositions and methods of this invention have been described interms of preferred embodiments, it will be apparent to those of skill inthe art that variations may be applied to the methods and in the stepsor in the sequence of steps of the method described herein withoutdeparting from the concept, spirit, and scope of the invention. Morespecifically, it will be apparent that certain agents that are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope, and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1.-31. (canceled)
 32. A method of cell replacement therapy for replacingcells that are known to be essentially non-dividing cells, the methodcomprising administering an effective amount of precursor cells that arenot pluripotent stem cells, the precursor cells comprising expressionvectors encoding a cell cycle-dependent promoter operably linked to asuicide gene coding sequence, wherein the suicide gene iscytomegalovirus (CMV) UL97 gene or mutant herpes simplex virus thymidinekinase (HSV-TK), and administering to the subject an amount of a prodrugthat is activated by the suicide gene, the prodrug being administered inan amount effective to eliminate cycling precursor cells.
 33. The methodof claim 32, wherein the precursor cells are neural precursor cells,cardiomyocyte precursor cells, or pancreatic precursor cells.
 34. Themethod of claim 32, wherein the subject is a mammal.
 35. The method ofclaim 34, wherein the mammal is a mouse, rat, non-human primate, orhuman.
 36. The method of claim 32, wherein the genome of the host cellcomprises a genome essentially identical to the genome of the subject.37. The method of claim 32, wherein the essentially non-dividing cellsto be replaced comprise dopaminergic cells and the host cells comprisedopaminergic neural precursor cells, defined as expressing tyrosinehydroxylase or dopamine active transporter.
 38. The method of claim 32,wherein the subject has Parkinson's disease.
 39. The method of claim 32,wherein the prodrug is penciclovir.
 40. The method of claim 32, whereinthe prodrug is administered more than once.
 41. The method of claim 32,wherein the prodrug is administered after a sufficient period of timefor the precursor cells to initiate differentiation.
 42. The method ofclaim 41, wherein the period of time is 3-6 days.
 43. The method ofclaim 41, wherein the period of time is 7-15 days.
 44. The method ofclaim 32, wherein the prodrug is administered by injection.
 45. Themethod of claim 32, wherein the precursor cells are further defined asneural precursor cells, cardiomyocyte precursor cells, endothelialprecursor cells, pancreatic precursor cells, kidney precursor cells,oligodendrocyte precursor cells, hematopoietic precursor cells, myeloidprecursor cells, mesenchymal precursor cells, retinal precursor cells,or osteoclast precursor cells.
 46. The method of claim 45, wherein theprecursor cells are further defined as a neural precursor cells.
 47. Themethod of claim 46, wherein the neural precursor cells are furtherdefined as expressing at least one of the markers selected from thegroup consisting of musashi, nestin, sox2, vimentin, pax6, and sox1. 48.The method of claim 32, wherein the CMV-UL97 is mutant CMV-UL97.
 49. Themethod of claim 32, wherein the mutant HSV-TK is SR11, SR26, SR39, SR4,SR15, SR32, or SR53.
 50. The method of claim 32, wherein the cellcycle-dependent promoter is a Ki-67, PCNA, CCNA2, CCNB2, DLGAPS, orTOP2A promoter.
 51. The method of claim 32, wherein the cellcycle-dependent promoter is a synthetic cell cycle promoter.
 52. Themethod of claim 32, wherein the expression vectors further encode aselectable marker.
 53. The method of claim 52, wherein the selectablemarker is encoded by an antibiotic resistance gene or a gene encoding afluorescent protein.
 54. The method of claim 32, wherein the vectors areviral vectors.
 55. The method of claim 54, wherein the viral vectors arelentiviral vectors, adenoviral vectors, retroviral vectors, vacciniaviral vectors, adeno-associated viral vectors, herpes viral vectors, orpolyoma viral vectors.